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E Lazarides

Publications and source records attributed to E Lazarides.

122 records · Page 7Linked to original sources
PubMed1975

Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells.

Antibodies specific for the skeletal muscle structural protein alpha-actinin are used to localize this protein by indirect immunofluorescence in nonmuscle cells. In cultured nonmuscle cells, alpha-actinin is localized along or between actin filament bundles producing an almost regular periodicity. The protein is also detected in the form of fluorescent plaques at some ends of actin filament bundles, as well as in a filamentous form in some overlap areas of cells. In spreading rat embryo cells, alpha-actinin assumes a focal distribution which corresponds to the vertices of a highly regular actin filament network. The results suggest that alpha-actinin may be involved in the organization of actin filament bundles, in the attachment of actin filaments to the plasma membrane, and in the assembly of actin filaments in areas of cell to cell contact.

Actinin↗
PubMed1975

Tropomyosin antibody: the specific localization of tropomyosin in nonmuscle cells.

An antibody against purified chicken skeletal muscle tropomyosin is used in indirect immunofluorescence to visualize the localization of tropomyosin in a variety of nonmuscle cells. The antibody produces a fluorescent pattern which is very similar to that obtained with an actin-specific antibody. This pattern is composed of fluorescent fibers which are shown to be coincident with the fibers seen with phase-contrast optics. High resolution epifluorescent microscopy reveals that fibers stained with the actin antibody show a continuous fluorescence, while fibers reacted with the tropomyosin antibody show a periodic fluorescence. Measurements indicate that the lengths of the fluorescent segments are variable with an average of 1.2 mum while the spacing between segments is approximately 0.4 mum.

Actins↗
PubMed1974

Actin is the naturally occurring inhibitor of deoxyribonuclease I.

Various tissues and cells in culture contain a specific inhibitor of DNase I (EC 3.1.4.5). In this paper evidence is presented that this inhibitor is actin, one of the major structural proteins of muscle and nonmuscle cells. (a) The inhibitor is a major cellular component constituting 5-10% of the soluble protein. (b) It migrates with actin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, having a characteristic molecular weight of 42,000. (c) It has an amino-acid composition closely similar to that of actin. (d) The peptide maps of the two proteins are nearly identical. (e) Skeletal muscle actin inhibits the enzymatic activity of DNase I. (f) DNase I-agarose affinity chromatography quantitatively retains purified skeletal muscle actin, and actin, specifically, from high-speed supernatants of whole cell extracts. (g) An antibody to purified inhibitor protein from calf thymus, used in indirect immunofluorescence on cells grown in culture, stains a two-dimensional network of fibers similar to that seen with an actin-specific antibody.The observation that actin can be isolated by DNase-agarose affinity chromatography provides a useful tool for the biochemical study of actin under different physiological conditions.

Actins↗
PubMed1974

Actin antibody: the specific visualization of actin filaments in non-muscle cells.

Actin purified from mouse fibroblasts by sodium dodecyl sulfate gel electrophoresis was used as antigen to obtain an antibody in rabbits. The elicited antibody was shown to be specific for actin as judged by immunodiffusion and complement fixation against partially purified mouse fibroblast actin and highly purified chicken muscle actin. The antibody was used in indirect immunofluorescence to demonstrate by fluorescence light microscopy the distribution and pattern of actin-containing filaments in a variety of cell types. Actin filaments were shown to span the cell length or to concentrate in "focal points" in patterns characteristic for each individual cell.

Actins↗
PubMed1971

Collagen polypeptides: normal release from polysomes in the absence of proline hydroxylation.

It is not necessary that proline be hydroxylated for the completion and release of nascent collagen chains from polysomes. Hydroxylation of collagen proline in vivo normally takes place predominantly on nascent polypeptides; however, in the presence of an inhibitor of hydroxylation, unhydroxylated chains are released. These chains may subsequently be hydroxylated when the inhibition is removed. The results clarify a controversy over the site of proline hydroxylation.

Animals↗
PubMedDate not supplied

Actin-containing microprocesses in the fusion of cultured chick myoblasts.

Scanning electron microscopic studies of myoblasts from 11- to 13-day-old chick embroyonic breast muscle cultured on collagen-coated glass coverslips showed six stages of development into multinucleated myotubes: (1) growth of flattened, spread-out cells for 20-30 hr following initiation of monolayer cultures; (2) extension of microprocesses (1-150 microM) from cells that have become spindle shaped; (3) contact and adherence of microprocesses from adjacent cells; (4) thickening of fused processes; (5) approximation of the cells; and (6) coalescence of the cells to form a spindle-shaped myotube. When the calcium-ion concentration in the growth medium was lowered--either by increasing the concentration of ethylene-glycol-bis(aminoethyl ether)N,N'-tetraacetate (EGTA)or by decreasing the cconcentration of free calciumion used--the number of microprocesses present on the cells was reduced. Presumably, however, these microprocesses could still fuse together, provided that the calciumion concentration was greater than 160 microM. Indirect immunofluorescence assay with actin-specific antibody indicated that actin is a major component of the myoblasts' microprocesses. Cytochalasin B (5 microgram/ml) caused the microprocesses to retract within 15 min and the myoblasts to round up and detach from the glass substrate. This was presumably caused by the action of the drug on actin filaments.

Actins↗
PubMedDate not supplied

Switching of subunit composition of muscle spectrin during myogenesis in vitro.

Spectrin comprises a family of polypeptides thought to be involved in mediating linkage of actin filaments to the plasma membrane in a wide variety of cell types (for reviews see refs 1-3). Spectrin is present as a tetramer composed of two non-identical subunits. Most cells express a common subunit with a molecular weight (Mr) of 240,000 (240 K; termed alpha-spectrin) in association with a polymorphic cell type-specific subunit: Mr 260 K in the intestinal terminal web (termed TW260), Mr 235 K in nervous tissue., liver, lymphocytes and fibroblasts (termed gamma-spectrin; also referred to as fodrin), and Mr 225/220 K in erythrocytes and adult cardiac and skeletal muscle (termed beta'- and beta-spectrin, respectively). We show here that primary chicken myoblasts express predominantly alpha gamma-spectrin, but on terminal differentiation in vitro the cells gradually switch to alpha beta-spectrin as a result of the onset of beta- and beta'-spectrin synthesis and by the subsequent differential stabilization of beta- and gamma-spectrin. This switching correlates with known changes in the biophysical properties and function of the developing muscle sarcolemma and cytoskeleton.

Animals↗
PubMedDate not supplied

beta-Spectrin limits alpha-spectrin assembly on membranes following synthesis in a chicken erythroid cell lysate.

Spectrin, the major protein of the subcortical actin network in erythrocytes, contains two non-identical subunits, alpha and beta (refs 1, 2). Spectrin is indirectly associated with the transmembrane anion transporter through the binding of beta-spectrin to an extrinsic protein, ankyrin. In chicken embryo erythroid cells, alpha-spectrin is synthesized in a threefold excess relative to beta-spectrin, although the two subunits are assembled in equimolar amounts. To investigate the regulation of assembly of equimolar amounts of spectrin, an in vitro system from chicken embryo erythroid cells has now been developed where synthesis and assembly of spectrin can be uncoupled and studied separately. Following the in vitro translation of threefold more alpha- than beta-spectrin, 95% of the beta-spectrin and equimolar amounts of alpha-spectrin bind post-translationally to spectrin-depleted rabbit red blood cell membranes, and the excess alpha-spectrin remains unbound. This alpha-spectrin cannot bind spectrin-depleted plasma membranes subsequently added to the lysate. The assembly of alpha-spectrin is, therefore, limited by the availability of beta-spectrin, and both subunits assemble post-translationally.

Animals↗
PubMedDate not supplied

Appearance of new variants of membrane skeletal protein 4.1 during terminal differentiation of avian erythroid and lenticular cells.

The erythrocyte plasma membrane is lined with a network of extrinsic proteins, mainly spectrin and actin, which constitute a reticulum tethered to the intrinsic anion transport protein of the lipid bilayer through a linker protein, ankyrin. Protein 4.1 forms a stable ternary complex with spectrin and actin, thereby strengthening the reticulum and anchoring it directly to the lipid bilayer or to another intrinsic protein, glycophorin. It has been found recently that spectrin, ankyrin and protein 4.1 are not erythrocyte-specific; this has elucidated further the mechanisms of plasma membrane assembly and modelling during the differentiation of diverse tissues. We have shown previously that protein 4.1 in chickens is most abundant in erythrocytes and lens cells, but is scarce or absent from other spectrin-rich cell types. In addition, it exists as a family of related polypeptides showing differential expression in these two tissues, suggesting variant-specific functions. Here we show that the pattern of protein 4.1 variants changes during the terminal differentiation of erythroid and lenticular cells, with novel variants appearing in postmitotic cells. The accumulation of these variants may lead to the final stabilization of the plasma membrane skeletons of these cells.

Animals↗
PubMedDate not supplied

Spectrin assembly in avian erythroid development is determined by competing reactions of subunit homo- and hetero-oligomerization.

Erythroid differentiation entails the biogenesis of a membrane skeleton, a network of proteins underlying and interacting with the plasma membrane, whose major constituent is the heterodimeric protein spectrin, composed of two structurally similar but distinct subunits, alpha (relative molecular mass (Mr) 240,000) and beta (Mr 220,000), which interact side-on with each other to form a long rod-like molecule. Interaction of this network with the membrane is mediated by the binding of the beta subunit to ankyrin, which in turn binds to the cytoplasmic domain of the transmembrane anion transporter (also referred to as band 3). Purified alpha and beta subunits of spectrin from the membrane of mature red blood cells will spontaneously heterodimerize, suggesting that assembly of the spectrin-actin skeleton is a simple self-assembly process, but in vivo studies with developing chicken embryo erythroid cells have indicated that assembly in vivo is more complex. We now present evidence that newly synthesized spectrin subunits in vivo or in vitro rapidly adopt one of two competing conformations, a heterodimer or a homo-oligomer. These competing reactions seem to determine the overall extent of spectrin assembled during erythroid development by determining which conformation will assemble onto the membrane-skeleton (the heterodimer) and which conformations are targeted for degradation (the homo-oligomers).

Animals↗