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.     

The lamprey (Lampetra fluviatilis) erythrocyte; morphology, ultrastructure, major plasma membrane proteins and phospholipids, and cytoskeletal organization.

The aim of this study was to characterize the erythrocyte of the lamprey (Lampetra fluviatilis), a primitive vertebrate. The lamprey erythrocyte predominantly has a non-axisymmetric stomatocytelike shape. It has a nucleus and a haemoglobin-filled cytosol with a few organelles and vesicular structures. Surprisingly, there is no marginal band of microtubules. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by Coomassie blue staining of isolated plasma membranes revealed a single band at the level of the human spectrin doublet. Major bands also occurred at approximately 175 kDa and comigrating with human erythrocyte actin (approximately 45 kDa). The presence of spectrin, actin and vimentin was shown by immunoblotting. Band 3 protein, the anion exchanger in higher vertebrates, seemed to be highly deficient or lacking, as was also the case with ankyrin. Confocal laser scanning microscopy combined with immunocytochemical methods showed spectrin, actin and vimentin mainly to be localized around the nucleus, from where actin- and vimentin-strands extended out into the cytoplasm. Actin also seemed to be present at the plasma membrane. Phospholipid analyses of plasma membrane preparations showed the presence of the same four major phospholipid groups as in the human erythrocyte, although with higher and lower amounts of phosphatidylcholine and sphingomyelin, respectively. The low fluorescein isothiocyanate conjugated annexin V binding, as monitored by flow cytometry, indicated that phosphatidylserine is mainly confined to the inner membrane leaflet in the lamprey erythrocyte plasma membrane.     

The lamprey (Lampetra fluviatilis) erythrocyte; morphology,ultrastructure, major plasma membrane proteins and phospholipids,and cytoskeletal organization

The aim of this study was to characterize the erythrocyte of the lamprey (Lampetra fluviatilis), a primitive vertebrate. The lamprey erythrocyte predominantly has a non-axisymmetric stomatocytelike shape. It has a nucleus and a haemoglobin-filled cytosol with a few organelles and vesicular structures. Surprisingly, there is no marginal band of microtubules. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by Coomassie blue staining of isolated plasma membranes revealed a single band at the level of the human spectrin doublet. Major bands also occurred at approximately 175 kDa and comigrating with human erythrocyte actin (~ 45 kDa). The presence of spectrin, actin and vimentin was shown by immunoblotting. Band 3 protein, the anion exchanger in higher vertebrates, seemed to be highly deficient or lacking, as was also the case with ankyrin. Confocal laser scanning microscopy combined with immunocytochemical methods showed spectrin, actin and vimentin mainly to be localized around the nucleus, from where actin- and vimentinstrands extended out into the cytoplasm. Actin also seemed to be present at the plasma membrane. Phospholipid analyses of plasma membrane preparations showed the presence of the same four major phospholipid groups as in the human erythrocyte, although with higher and lower amounts of phospatidylcholine and sphingomyelin, respectively. The low fluorescein isothiocyanate conjugated annexin V binding, as monitored by flow cytometry, indicated that phosphatidylserine is mainly confined to the inner membrane leaflet in the lamprey erythrocyte plasma membrane.     

Cell type-specific association between two types of spectrin and two types of intermediate filaments

We have demonstrated a differential association between two types of spectrin, from erythrocytes and brain, with two types of intermediate filaments, vimentin filaments and neurofilaments. Electron microscopy showed that erythrocyte spectrin promoted the binding of vimentin filaments to red cell inside-out vesicles via lateral associations with the filaments. In vitro binding studies showed that the association of spectrin with vimentin filaments was apparently saturable, increased with temperature, and could be prevented by heat denaturation of the spectrin. Comparisons were made between erythrocyte and brain spectrin binding to both vimentin filaments and neurofilaments. We found that vimentin filaments bound more erythrocyte spectrin than brain spectrin, while neurofilaments bound more brain spectrin than erythrocyte spectrin. Our results show that both erythroid and nonerythroid spectrins are capable of binding to intermediate filaments and that such associations may be characterized by differential affinities of the various types of spectrin with the several classes of intermediate filaments present in cells. Our results also suggest a role for both erythroid and nonerythroid spectrins in mediating the association of intermediate filaments with plasma membranes or other cytoskeletal elements.     

Crosslinking of membrane proteins during erythrocyte ageing

Membrane proteins of bovine erythrocytes were crosslinked with cupric di(1,10-phenanthroline) and analysed by one-dimensional and two-dimensional SDS-polyacrylamide gel electrophoresis. An increase in crosslinking of the Band 3 protein and of spectrin was found with increasing erythrocyte age suggesting an increased aggregation of main membrane proteins in aged erythrocytes.     

The lens membrane skeleton contains structures preferentially enriched in spectrin-actin or tropomodulin-actin complexes

The spectrin-based membrane skeleton plays an important role in determining the distributions and densities of receptors, ion channels, and pumps, thus influencing cell shape and deformability, cell polarity, and adhesion. In the paradigmatic human erythrocyte, short tropomodulin-capped actin filaments are cross-linked by spectrin into a hexagonal network, yet the extent to which this type of actin filament organization is utilized in the membrane skeletons of nonerythroid cells is not known. Here, we show that associations of tropomodulin and spectrin with actin in bovine lens fiber cells are distinct from that of the erythrocyte and imply a very different molecular organization. Mechanical disruption of the lens fiber cell membrane skeleton releases tropomodulin and actin-containing oligomeric complexes that can be isolated by gel filtration column chromatography, sucrose gradient centrifugation and immunoadsorption. These tropomodulin-actin complexes do not contain spectrin. Instead, spectrin is associated with actin in different complexes that do not contain tropomodulin. Immunofluorescence staining of isolated fiber cells further demonstrates that tropomodulin does not precisely colocalize with spectrin along the lateral membranes of lens fiber cells. Taken together, our data suggest that tropomodulin-capped actin filaments and spectrin-cross-linked actin filaments are assembled in distinct structures in the lens fiber cell membrane skeleton, indicating that it is organized quite differently from that of the erythrocyte membrane skeleton.     

Hemin-mediated dissociation of erythrocyte membrane skeletal proteins

Spectrin tetramers and oligomers in normal erythrocytes are cross-linked by actin and protein 4.1 to form a two-dimensional membrane skeletal network. In the present study, we find that hemin, a breakdown product of hemoglobin, progressively (a) alters the conformation of spectrin as revealed by electron microscope studies and by the decreased resistance of spectrin to proteolytic degradation, (b) alters the conformation of protein 4.1 as revealed by the increased mobility of protein 4.1 on nondenaturing gel electrophoresis, (c) weakens spectrin dimer alpha beta-dimer alpha beta, spectrin alpha-spectrin beta, as well as spectrin-protein 4.1 associations as analyzed by nondenaturing gel electrophoresis, and (d) diminishes the structural stability of erythrocyte membrane skeletons (i.e. Triton-insoluble ghost residues) subjected to mechanical shearing. Since hemin may be liberated from oxidized or unstable mutant hemoglobin under pathological conditions, these hemin-induced effects on spectrin, protein 4.1, and membrane skeletal stability may play a role in the membrane lesion of these erythrocytes.     

Widespread occurrence of avian spectrin in nonerythroid cells

We have prepared an antibody against chicken erythrocyte α spectrin, using as immunogen protein purified by two-dimensional polyacrylamide gel electrophoresis. One- and two-dimensional immunoautoradiography show that this antiserum reacts only with α spectrin in chicken erythrocytes and crossreacts with α 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.     

Association of spectrin with desmin intermediate filaments

The association of erythrocyte spectrin with desmin filaments was investigated using two in vitro assays. The ability of spectrin to promote the interaction of desmin filaments with membranes was investigated by electron microscopy of desmin filament-erythrocyte inside-out vesicle preparations. Desmin filaments bound to erythrocyte inside-out vesicles in a spectrin-dependent manner, demonstrating that spectrin is capable of mediating the association of desmin filaments with plasma membranes. A quantitative sedimentation assay was used to demonstrate the direct association of spectrin with desmin filaments in vitro. When increasing concentrations of spectrin were incubated with desmin filaments, spectrin cosedimented with desmin filaments in a concentration-dependent manner. At near saturation the spectrin:desmin molar ratio in the sedimented complex was 1:230. Our results suggest that, in addition to its well characterized associations with actin, spectrin functions to mediate the association of intermediate filaments with plasma membranes. It might be that nonerythrocyte spectrins share erythrocyte spectrin's ability to bind to intermediate filaments and function in nonerythroid cells to promote the interaction of intermediate filaments with actin filaments and/or the plasma membrane.     

INTRAMEMBRANE PARTICLE AGGREGATION IN ERYTHROCYTE GHOSTS : I. The Effects of Protein Removal

We have used freeze-etching and SDS-polyacrylamide gel electrophoresis to study the conditions under which the intramembrane particles of the human erythrocyte ghost may be aggregated. The fibrous membrane protein, spectrin, can be almost entirely removed from erythrocyte ghosts with little or no change in the distribution of the particles. However, after spectrin depletion, particle aggregation in the plane of the membrane may be induced by conditions which cause little aggregation in freshly prepared ghosts. This suggests that the spectrin molecules form a molecular meshwork which limits the translational mobility of the erythrocyte membrane particles.     

Alteration of rheological properties of human erythrocytes by crosslinking of membrane proteins

The crosslinking of membrane proteins of human erythrocytes by diamide (diazene dicarboxylic acid bis(N,N-dimethylamide) ) was quantified by 4% polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. The relation between the crosslinking of membrane proteins and erythrocyte functions (rheological and oxygen transporting) was quantitatively examined. (i) The crosslinking of membrane protein was induced by diamide, without changing the shape and the contents of intracellular organic phosphates (adenylates and 2,3-diphosphoglycerate). The intensity of spectrin 2 in SDS-polyacrylamide gel electrophoresis decreased proportionally to diamide concentration. The percentage decrease in spectrin 2 (using band 3 as an internal standard) was the most appropriate indicator for crosslinking ("% crosslinking'). (ii) The suspension viscosity of erythrocytes increased in proportion to the percentage of crosslinking, in the range of applied shear rates of 3.76-752 s-1. (iii) Erythrocyte deformability (measured by a high-shear rheoscope) was reduced by the crosslinking. The change was detectable even at 5% crosslinking. (iv) Rouleaux formation (measured by a television image analyzer combined with a low-shear rheoscope) was inhibited by the crosslinking. The inhibition was also sensitively detected at more than 5% crosslinking. (v) Hemoglobin in erythrocytes was chemically modified by higher dose of diamide (probably by the binding of diamide with sulfhydryl groups). Also the oxygen affinity of hemoglobin increased and the heme-heme interaction decreased. (vi) The reduction of the crosslinking of membrane proteins by dithiothreitol apparently reversed the intensity of spectrin bands in SDS-polyacrylamide gel electrophoresis and the erythrocyte functions (the suspension viscosity and the deformability), though not completely.     

Characteristics of membrane protein phosphorylation in Plasmodium berghei-infected mouse erythrocytes

Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (32P)orthophosphate and incubating isolated membrane with (gamma-32P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with Mr 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

Actin in erythrocyte ghosts and its association with spectrin. Evidence for a nonfilamentous form of these two molecules in situ

Actin was isolated from erythrocyte ghosts. It is identical to muscle actin in its molecular weight, net charge, ability to polymerize into filaments with the double helical morphology, and its decoration with heavy meromyosin (HMM). when erythrocyte ghosts are incubated in 0.1 mM EDTA, actin and spectrin are solubilized. Spectrin has a larger molecular weight than muscle myosin. When salt is added to the EDTA extract, a branching filamentous polymer is formed. However, when muscle actin and the EDTA extract are mixed together in the presence of salt, the viscosity achieved is less than the viscosity of the solution if spectrin is omitted. Thus, spectrin seems to inhibit the polymerization of actin. If the actin is already polymerized, the addition of spectrin increases the viscosity of the solution, presumably by cross-linking the actin filaments. The addition of HMM of trypsin to erythrocyte ghosts results in filament formation in situ. These agents apparently act by detaching erythrocyte actin from spectrin, thereby allowing the polmerization of one or both proteins to occur. Since filaments are not present in untreated erythrocyte ghosts, we conclude that erythrocyte actin and spectrin associate to form an anastomosing network beneath the erythrocyte membrane. This network presumably functions in restricting the lateral movement of membrane-penetrating particles.     

Spectrin-actin associations studied by electron microscopy of shadowed preparations

By shadowing specimens dried onto mica sheets we have obtained clear images of actin crosslinked by spectrin, an actin-binding protein found in erythrocytes. We conclude that spectrin dimers possess a single binding site for F actin. Tetramers formed by head-to-head association of two dimers possess two actin binding sites, one at each tail. Polymerizing G actin in the presence of spectrin tetramers or mixing preformed F actin with spectrin tetramer plus band 4.1 results in an extensively crosslinked network of actin filaments. When G actin is polymerized in the presence of spectrin at spectrin:actin mole ratios close to that present on the erythrocyte membrane, large amorphous protein networks are formed. These networks are clusters of spectrin around 25 nm diameter structures which may be actin protofilaments. These networks are similar to the cytoskeletal network seen after erythrocyte membranes are extracted with detergent, and may represent the first in vitro assembly of a cytoskeletal complex resembling that of the native cell both biochemically and structurally.     

Analysis of erythrocyte membrane proteins from dystrophic hamsters

To search for potentially mutant proteins, we have investigated erythrocyte ghost proteins from normal and dystrophic hamster by two-dimensional gel electrophoresis. No significant differences are observed between dystrophic and normal erythrocytes in their peptide patterns on SDS-polyacrylamide gel electrophoresis while on two-dimensional gels a protein spot of approximate Mr 20 000 with an approximate isoelectric point of 4.5 is found in erythrocytes from dystrophic animals and is consistently absent in normal erythrocytes. A large population of erythrocyte (60%) from dystrophic hamsters shows distorted shape as visualized by scanning electron microscopy. The nature of this protein and its relevance in hamster muscular dystrophy are at present not known.     

Characteristics of Membrane Protein Phosphorylation in Plasmodium berghei-Infected Mouse Erythrocytes1

ABSTRACT. Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (³²P)orthophosphate and incubating isolated membrane with (γ-³²P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with M, 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

The spectrin-actin junction of erythrocyte membrane skeletons

High-resolution electron microscopy of erythrocyte membrane skeletons has provided striking images of a regular lattice-like organization with five or six spectrin molecules attached to short actin filaments to form a sheet of five- and six-sided polygons. Visualization of the membrane skeletons has focused attention on the (spectrin)5,6-actin oligomers, which form the vertices of the polygons, as basic structural units of the lattice. Membrane skeletons and isolated junctional complexes contain four proteins that are stable components of this structure in the following ratios: 1 mol of spectrin dimer, 2-3 mol of actin, 1 mol of protein 4.1 and 0.1-0.5 mol of protein 4.9 (numbers refer to mobility on SDS gels). Additional proteins have been identified that are candidates to interact with the junction, based on in vitro assays, although they have not yet been localized to this structure and include: tropomyosin, tropomyosin-binding protein and adducin. The spectrin-actin complex with its associated proteins has a key structural role in mediating cross-linking of spectrin into the network of the membrane skeleton, and is a potential site for regulation of membrane properties. The purpose of this article is to review properties of known and potential constituent proteins of the spectrin-actin junction, regulation of their interactions, the role of junction proteins in erythrocyte membrane dysfunction, and to consider aspects of assembly of the junctions.     

Immunodetection of spectrin-like proteins in yeasts

Spectrin, a component of the membrane skeleton in erythrocytes and other animal cells, has also been identified in plant and fungal cells. However, its postulated role, i.e., the maintenance of shape and elasticity of the plasma membrane, is probably not exerted in walled cells. To study spectrin in these cells, we chose yeasts because of a high morphological variability of their life cycle. The localization of spectrin in the cells and protoplasts of Saccharomyces cerevisiae and Schizosaccharomyces japonicus var. versatilis was detected by immunoblotting, indirect immunofluorescence, and immunogold electron microscopy techniques with the use of anti-chicken and anti-human erythrocyte spectrin antibodies. A protein band of 220-240 kDa and some bands of lower relative mass were detected in cell and protoplast extracts of both yeast strains. Spectrin-like proteins were revealed by fluorescence microscopy at cell surfaces and in vacuolar membranes. Immunogold-labelling showed spectrin-like proteins in the plasma membrane, endoplasmic reticulum, vacuoles, nuclei, vesicles, mitochondria, and cell walls. The topology of spectrin was not affected by actin depolymerization with Latrunculin B nor was it changed in either act1-1 or cdc42 mutants, under restrictive conditions. Under osmotic stress, both spectrin and actin were delocalized and appeared in the form of large clusters in the cytoplasm. It is concluded that a protein cross-reacting with spectrin antibodies is present in fission and budding yeasts. Generally, it is located in the proximity of the plasma membrane and other intracellular membranes, probably as a part of the membrane skeleton. No evidence of its relationship to either actin or growth zones of the cell can be provided.     

Vimentin filaments in spreading,randomly locomoting,and f-met-leu-phe-treated neutrophils

Summary Human neutrophils contain intermediate filaments of the vimentin type. A cytoskeletal preparation, produced by high-salt and Triton X-100 extraction of human neutrophils, reveals a major band at 57000 M _r that comigrates with 3T3 cell vimentin on one-dimensional gels. Two-dimensional gel electrophoresis of whole neutrophils illustrates the presence of vimentin but not desminor keratin-filament subunits. The presence of vimentin in neutrophils is also shown by its specific staining with avian vimentin antiserum by two-dimensional gel immunoautoradiography. Indirect immunofluorescence studies show that vimentin antiserum labels an area on one side of the nucleus in spreading neutrophils. This bright area appears as a loose knot of vimentin filaments; a few filaments may radiate from the knot. In contrast to spreading neutrophils, those undergoing random locomotion contain a fine network of filaments that are located in the cytoplasm between the nucleus and the trailing end of the cell. Similarly, in chemoattractant-treated neutrophils, vimentin filaments are bundled in the uropod. Transmission electron microscopy of human neutrophil monolayers confirms the intracellular distribution of intermediate filaments as shown by immunofluorescence in spreading and randomly locomoting cells.     

Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins

I have purified a high molecular weight actin filament gelation protein (GP-260) from Acanthamoeba castellanii, and found by immunological cross-reactivity that it is related to vertebrate spectrins, but not to two other high molecular weight actin-binding proteins, filamin or the microtubule-associated protein, MAP-2. GP-260 was purified by chromatography on DEAE-cellulose, selective precipitation with actin and myosin-II, chromatography on hydroxylapatite in 0.6 M Kl, and selective precipitation at low ionic strength. The yield was 1-2 micrograms/g cells. GP-260 had the same electrophoretic mobility in SDS as the 260,000-mol-wt alpha-chain of spectrin from pig erythrocytes and brain. Electron micrographs of GP-260 shadowed on mica showed slender rod-shaped particles 80-110 nm long. GP-260 raised the low shear apparent viscosity of solutions of Acanthamoeba actin filaments and, at 100 micrograms/ml, formed a gel with a 8 microM actin. Purified antibodies to GP-260 reacted with both 260,000- and 240,000-mol-wt polypeptides in samples of whole ameba proteins separated by gel electrophoresis in SDS, but only the 260,000-mol-wt polypeptide was extracted from the cell with 0.34 M sucrose and purified in this study. These antibodies to GP-260 also reacted with purified spectrin from pig brain and erythrocytes, and antibodies to human erythrocyte spectrin bound to GP-260 and the 240,000-mol-wt polypeptide present in the whole ameba. The antibodies to GP-260 did not bind to chicken gizzard filamin or pig brain MAP-2, but they did react with high molecular weight polypeptides from man, a marsupial, a fish, a clam, a myxomycete, and two other amebas. Fluorescent antibody staining with purified antibodies to GP-260 showed that it is concentrated near the plasma membrane in the ameba.