Domain analysis of supervillin, an F-actin bundling plasma membrane protein with functional nuclear localization signals.

A growing number of actin-associated membrane proteins have been implicated in motile processes, adhesive interactions, and signal transduction to the cell nucleus. We report here that supervillin, an F-actin binding protein originally isolated from bovine neutrophil plasma membranes, contains functional nuclear targeting signals and localizes at or near vinculin-containing focal adhesion plaques in COS7-2 and CV1 cells. Overexpression of full-length supervillin in these cells disrupts the integrity of focal adhesion plaques and results in increased levels of F-actin and vinculin. Localization studies of chimeric proteins containing supervillin sequences fused with the enhanced green fluorescent protein indicate that: (1) the amino terminus promotes F-actin binding, targeting to focal adhesions, and limited nuclear localization; (2) the dominant nuclear targeting signal is in the center of the protein; and (3) the carboxy-terminal villin/gelsolin homology domain of supervillin does not, by itself, bind tightly to the actin cytoskeleton in vivo. Overexpression of chimeras containing both the amino-terminal F-actin binding site(s) and the dominant nuclear targeting signal results in the formation of large nuclear bundles containing F-actin, supervillin, and lamin. These results suggest that supervillin may contribute to cytoarchitecture in the nucleus, as well as at the plasma membrane.

Cloning, characterization, and chromosomal localization of human superillin (SVIL).

Supervillin is a 205-kDa F-actin binding protein originally isolated from bovine neutrophils. This protein is tightly associated with both actin filaments and plasma membranes, suggesting that it forms a high-affinity link between the actin cytoskeleton and the membrane. Human supervillin cDNAs cloned from normal human kidney and from the cervical carcinoma HeLa S3 predict a bipartite structure with three potential nuclear localization signals in the NH2-terminus and three potential actin-binding sequences in the COOH-terminus. In fact, throughout its length, the COOH-terminal half of supervillin is similar to segments 2-6 plus the COOH-terminal "headpiece" of villin, an actin-binding protein in intestinal microvilli. A comparison of the bovine and human sequences indicates that supervillin is highly conserved at the amino acid level, with 79.2% identity of the NH2-terminus and conservation of three of the four nuclear localization signals found in bovine supervillin. The COOH-terminus is even more conserved, with 95.1% amino acid identity overall and 100% conservation of the villin-like headpiece. Supervillin mRNAs are expressed in all human tissue tested, bu are most abundant in muscle, bone marrow, thyroid gland, and salivary gland; comparatively little message is found in brain. Human supervillin mRNA is approximately 7.5 kb; this message is especially abundant in HeLa S3 cervical carcinoma, SW480 adenocarcinoma, and A549 lung carcinoma cell lines. The human supervillin gene (SVIL) is localized to a single chromosomal locus at 10p11.2, a region that is deleted in some prostate tumors.

Actin-binding membrane proteins identified by F-actin blot overlays.

Actin and associated proteins at the cytoskeleton-plasma membrane interface stabilize the membrane bilayer, control cell shape, and delimit specialized membrane domains. To identify membrane proteins that bind directly to F-actin, we have developed a blot overlay assay with 125I-labeled F-actin. In the soil amoebae, Dictyostelium discoideum, the major proteins reactive in this assay are p30a, a 34-kD peripheral membrane protein that is concentrated in filopodia and at sites of cell-cell adhesion, and ponticulin, a 17-kD transmembrane glycoprotein required for efficient chemotaxis and for control of pseudopod dynamics. Proteins with apparent molecular masses of approximately 34- and approximately 17-kD also are observed on F-actin blot overlays of many mammalian cell lines. However, in mammalian cells, the most prominent F-actin binding proteins in this assay exhibit apparent molecular masses of 78-, 80-, 81-, approximately 120-, and 205-kD. Bovine neutrophils contain the 78-, 81-, and 205-kD proteins, all of which co-isolate with a plasma membrane-enriched fraction. We have previously identified the 78-, 80-, and 81-kD proteins as moesin, radixin, and ezrin, respectively. These proteins, which are members of the protein 4.1 superfamily, colocalize with actin in cell surface extensions and have been implicated in the protrusion of microvilli, filopodia, and membrane ruffles. The 205-kD protein (p205) appears to be absent from current databases, and its characteristics are still under investigation. We here report that the 120-kD protein is drebrin, a submembranous actin-binding protein originally identified as a developmentally regulated brain protein. Thus, it appears that F-actin blot overlays provide an efficient assay for simultaneous monitoring of a subset of F-actin binding proteins, including p30a, ponticulin, moesin, radixin, ezrin, p205, and drebrin.

Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily.

Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

Effect of antibodies to membrane skeletal proteins on the shape of erythrocytes and their ability to respond to shape-modulating agents. Important role of 4.1 protein in the determination/maintenance of the discoid shape of erythrocytes.

Monospecific anti-spectrin, anti-4.1 protein, anti-ankyrin, and anti-band 3 cytoplasmic domain antibodies were individually internalized in human erythrocytes by hypotonic lysis in the presence of 2 mM Mg-ATP. The concentrations of the polyclonal antibodies, or their Fab fragments, varied from 0.5 to 4.0 mg/ml. After resealing and glutaraldehyde fixation, the shape of the erythrocytes was examined by phase-contrast or scanning electron microscopy. The antibody-internalized cells were also treated, prior to fixation, with dinitrophenol, chlorpromazine (membrane-penetrable crenation- and cup-forming agents, respectively), or serum albumin (membrane nonpenetrating cup-forming agent). Anti-spectrin antibodies produced a cup shape in the cells at low concentrations (0.5 mg/ml), with a few cells showing multiple cavities. The proportion of the latter increased with increasing concentration of the antibody. The Fab fragments of the antibody were without any effect on the shape. At lower concentrations of anti-4.1 antibodies (0.5 and 1 mg/ml), crenations were observed in antibody-internalized cells. At higher antibody concentrations, the cells underwent sphering. These looked dense under the phase-contrast microscope and often were found to project vesicles from their surface via long stalks. These were generally absent on cells when viewed under the electron microscope, probably due to their breakage during processing. The Fab fragments of the anti-4.1, at equimolar concentrations, were as effective as the intact antibody in altering the cell shape. Anti-ankyrin antibodies or the Fab fragments produced no effect on the shape of the cell. The intact anti-band 3 cytoplasmic domain antibodies, but not their Fab fragments, produced cups at low concentrations and multiple cavities at higher concentrations and multiple cavities at higher concentrations. The anti-spectrin- and anti-band 3 cytoplasmic domain-internalized cells resisted the shape-modulating actions of dinitrophenol, chlorpromazine, and serum albumin. The anti-4.1 antibody-internalized cells also did not respond to dinitrophenol and serum albumin; however, they were converted to contracted cups in the presence of high concentration of chlorpromazine. The cells in which anti-ankyrin antibodies were internalized responded to the actions of the shape-changing agents similarly to normal cells. The results show that the 4.1 protein plays an important role in the determination or maintenance of the normal shape of the erythrocyte and that the cells possessing antibody-modified membrane skeleton do not respond to amphiphilic ions in the manner that normal cells do.

Neutral polymers elicit, and antibodies to spectrin, band 4.1 protein and cytoplasmic domain of band 3 protein inhibit the concanavalin A-mediated agglutination of human erythrocytes.

Concanavalin A (Con A) is known to agglutinate human erythrocytes if the cells are pre-treated with a proteinase or neuraminidase. We report that untreated cells can also be made to agglutinate with the lectin if the lectin-bound cells are treated with anti-Con A antibodies, or if a neutral polymer such as serum albumin, polyvinylpyrrolidone or Ficoll is added. Thus, Con A falls in the category of 'incomplete' lectins. The polymer induces Con A-agglutinability without altering the receptor number, or deformability of the cells. If the polymer is sequestered within erythrocyte ghosts, Con A is unable to agglutinate them; but the presence of the polymer only on the outer surface (as in intact cells) or on both the surfaces permits agglutinability. Thus, the site of the polymer effect resides on the outer surface of the membrane. The polymer, however, is unable to induce agglutinability in erythrocyte vesicles, whose membrane lacks skeletal proteins. The result suggests a positive role for the membrane skeleton in the process of agglutination brought about by the polymer, as is true also for the agglutination of proteinase-treated cells. In order to obtain detailed information on the proteins participating in agglutination, monospecific antibodies to spectrins, band 4.1 protein, ankyrin and the cytoplasmic domain of band 3 protein were internalized in erythrocytes. It is found that anti-spectrin and anti-band 3 cytoplasmic domain, but not their Fab's, inhibit the Con A-mediated agglutinability partially, and anti-4.1 antibodies, as well as the Fab's, inhibit the agglutinability substantially. Anti-ankyrin, however, was without any effect. The results confirm a positive role for the membrane skeleton in the Con A-mediated agglutination of normal erythrocytes in the presence of a neutral polymer, or in proteinase treated cells. We also provide evidence for requirement of Mg-ATP in the agglutination process.

Relationship between the concanavalin A-agglutinability and deformability of human erythrocytes.

Cellular deformability has been proposed in the past as a major determinant of lectin-mediated agglutination of cells. In this paper we have evaluated the correlation between deformability and Con A-agglutinability of human erythrocytes by subjecting them to agents that alter either one of the properties and evaluating the effect on the other property. The following results have been obtained: (i) Treatment with pronase or trypsin, which makes the Con A-nonagglutinable normal red cells highly agglutinable, has practically no effect on deformability; while neuraminidase treatment, with a similar effect on agglutinability, produces a small but statistically significant reduction in deformability. (ii) Diamide treatment, on the other hand, produces a drastic reduction in the deformability of pronase-treated erythrocytes but has no effect on the Con A-agglutinability of the cells. Dinitrophenol also reduces deformability but without altering the agglutinability, (iii) Chlorpromazine, at 2 x 10(-5) M, does not have any effect on the deformability of trypsinized cells, but increases the agglutinability substantially. When the Con A-agglutinability of the cells and their deformability after these treatments are compared, a correlation coefficient r = -0.353 (P greater than 0.1) is obtained. This indicates the lack of any direct correlation between the two parameters, and rules out any significant role of deformability in the determination of Con A-agglutinability of erythrocytes. The agglutination with the lectin is completely reversed by methyl alpha-D-mannoside, the specific inhibitory sugar for Con A, also ruling out any secondary role for deformability in the non-lectin-mediated stabilization of clumps. Upon incubation of normal erythrocytes with Con A. a dose-dependent decrease in deformability is observed, with the deformability index falling to almost 25% of the normal value with 500 microgram/ml Con A. This indicates that Con A binding to its receptor produces changes in the membrane probably by altering properties of the membrane skeleton.

The role of the membrane skeleton in concanavalin A-mediated agglutination of human erythrocytes.

In the erythrocyte membrane, the mobility of band 3 protein, the receptor for concanavalin A (Con A), is drastically reduced by the membrane skeleton. Yet, the vesicles free of membrane skeletal proteins, isolated from the highly agglutinable proteinase-treated cells, are found to be devoid of Con A agglutinability. The vesicles bind Con A in normal amounts, and remain agglutinable with the wheat germ and Ricinus agglutinins. Intracellular entrapment of monospecific antibodies to spectrin and 4.1 protein (two of the major skeletal components of the membrane) is also found to inhibit agglutination by 30-50%. Thus the membrane skeleton appears to play a positive role in the agglutination of the cells with Con A. The anti-ankyrin antibodies are found to be without any effect. The anti-band 3 (cytoplasmic domain) antibodies are also inhibitory to agglutination. Since Con A binding to cells alters the shape responses and deformability of the cells, and the cells resist fragmentation at 49 degrees C, the properties of the whole skeleton, especially spectrin, appear to be changed. The Con A-bound membranes also do not release the complex of spectrin-band 4.1-actin when extracted with a hypotonic medium. It appears that Con A binding leads to interaction of the cytoplasmic domain of the receptor with a skeletal component, possibly spectrin. Subsequent to this, the receptor molecules and the skeletal proteins undergo aggregation in the membrane, which is detected by their crosslinking by an 8.6-A span bifunctional reagent. The contractility believed to be associated with the membrane skeleton may be responsible for the aggregation.

Effect of ethanol on turpentine-induced acute phase response in rats.

Turpentine-induced acute-phase response and its modulation by ethanol in rats at 48 hr has been studied. There was more than 2.3-5.1 fold increase in fibrinogen and seromucoids concentrations in plasma, accompanied by 28% decline in albumin concentration in turpentine-stimulated rats. The fractional synthesis rate of these two acute-phase proteins was increased by 4.1-6.4 fold, while that of albumin (non acute-phase protein) was reduced by 32.6%. Ethanol inhibited this induction of acute-phase protein synthesis at 48 hr. The inhibition of acute-phase response by ethanol was significantly more pronounced for seromucoids than for fibrinogen and appeared to be dependent on the carbohydrate content of the acute-phase glycoprotein.

Effect of chronic ethanol ingestion on liver and plasma protein synthesis in galactosamine-induced hepatitis in rats.

In rats a single injection (330 mg/kg) of D-galactosamine significantly inhibited in vivo incorporation of 14C-amino acids into fibrinogen, seromucoids and total liver proteins, but not albumin, by one third. Prior chronic exposure to ethanol (5 g/kg/day for 6 weeks) potentiated the inhibition of synthesis of all protein fractions (including albumin) up to about 50%. Propylthiouracil had no beneficial but deleterious effect against this potentiation of hepatotoxicity.