Enucleation eliminates a differentiation-specific surface marker from normal and leukemic murine erythroid cells

Mammalian erythropoiesis includes a step in which the nucleus is extruded through the cell membrane. We have investigated the relationship between concanavalinA (conA) plasma membrane receptors, which are known to leave the incipient reticulocyte during enucleation, and regions of the plasma membrane which bind merocyanine 540, a differentiation-specific marker of hematopoietic cells. The distribution of these two fluorescent probes was examined on living cells from the spleens of neonatal mice and on erythroleukemia cells induced to enucleate in culture. In both cases, the region of the membrane extruded with the nucleus preferentially binds conA and merocyanine 540, whereas the plasma membrane which is left behind retains the capacity to bind another lectin, wheat germ agglutinin (WGA). The implications of these findings are discussed with respect to the mechanism by which markers are eliminated from the erythrocyte cell surface.     

Alterations in plasma membrane lipid organization during lymphocyte differentiation

The fluorescent probe merocyanine 540, which binds preferentially to bilayers in which the lipids are loosely packed, was used to investigate changes in the organization of the lipids of the lymphocyte plasma membrane during primary and secondary lymphopoiesis. When mouse thymocytes were incubated with the dye, most immature cells stained, while most mature cells, about to enter the peripheral circulation, did not. Similarly, mature lymphocytes from both mouse and human peripheral blood did not stain, but these same cells did when activated by in vitro mitogenic stimulation. Freshly isolated splenic lymphocytes, presumably activated in vivo by antigen, also bound merocyanine 540, but after 48 hours of culture in the absence of stimulus they displayed only a low affinity for the dye, a phenotype that reverted to a high affinity upon mitogenic stimulation. These results suggest that changes in the organization of the lipids of the plasma membrane take place during lymphocyte differentiation: viz., immature cells possess a disordered membrane that becomes increasingly ordered as the cells mature and enter the peripheral circulation; then, upon antigen-induced differentiation, the plasma membrane again becomes disordered. These lipid organization changes are discussed in the context of their possible role in the regulation of lymphocyte circulation via intercellular interactions between lymphocytes and cells of the reticuloendothelial system.     

Effects of local anesthetics on membrane properties II. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins

Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface.     

Effects of local anesthetics on membrane properties. II. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins.

Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell.

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

Changes in the organization of the lipid bilayer of the plasma membrane during spermatogenesis and epididymal maturation

Ram, bull, and mouse sperm cells were stained with several fluorescent membrane probes. In contrast to nonspecific probes, merocyanine 540 (MC540), which displays preferential binding to loosely packed phospholipids in model membranes, was specifically localized to the anterior portion of the head and the midpiece of mature sperm. To establish when during development this distinctive staining pattern was acquired, germ cells from prepubescent and adult mouse testes as well as sperm from the caput, corpus, and cauda epididymides were isolated and examined. Localized staining with MC540 was not observed until sperm reached the corpus epididymidis, where those cells with a completely translocated (i.e., distally located) cytoplasmic droplet fluoresced. Likewise, when sperm were stained with fluoresceinated concanavalin A (fl-ConA), a localized pattern of fluorescence with lectin restricted to the anterior portion of the head was not observed until the corpus epididymidis was reached. However, in contrast to MC540 staining, only a fraction of sperm with completely translocated droplets exhibited this localized staining with fl-ConA, the remainder exhibiting diffuse fluorescence over the entire cell as seen on caput epididymal sperm. These developmental changes in staining patterns are specific to murine cells, since no change in the pattern of staining by either MC540 or fl-ConA was seen on epididymal sperm of the ram. These results are discussed with respect to: 1) species-to-species differences in sperm membrane features; and 2) the hypothesis that domains of loosely packed lipids may be involved in the regionalization of membrane proteins that occurs during sperm development.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various various lectins is Ricinuscommunis > wheat germ concanavalin A soybean >Limuluspolyphemus. No agglutination was noted for Ulex europaeus. Using ¹²⁵I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites as sites for concanavalin A and soybean lectins. Sodium deoxy-cholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity colums. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

Plasma membrane lipid organization and the adherence of differentiating lymphocytes to macrophages

Changes in the packing of phospholipids in the plasma membrane of lymphocytes occur during differentiation within primary and secondary lymphoid organs. As they differentiate, lymphocytes interact with a variety of reticuloendothelial cells, including macrophages. To investigate a possible relation between these two phenomena, the strength of the interactions between lymphocytes and macrophages was measured in vitro as a function of the tightness of packing of phospholipids on the lymphocyte surface. Strength of adherence was measured by the ability of lymphocytes to remain adherent to macrophages when subjected to increasing centrifugal forces. Phospholipid packing was assessed using the fluorescent lipophilic probe merocyanine 540 (MC540), which preferentially binds to bilayers in which the lipids are more loosely packed. Three subpopulations of murine thymocytes were resolved with respect to strength of adherence to peritoneal or thymic macrophages. To determine whether these subpopulations corresponded with the three classes of cells distinguishable by MC540 fluorescence, populations enriched for staining or non-staining cells, and cells sorted on the basis of MC540 fluorescence intensity, were examined. The least fluorescent cells were the least strongly adherent; the most fluorescent cells were the most strongly adherent; and cells of intermediate fluorescence had intermediate adherence. When splenic lymphocytes were examined with respect to adherence to peritoneal or splenic macrophages, similar patterns of fluorescence and adherence were seen. These results suggest that the organization of the plasma membrane lipid bilayer of lymphocytes may be involved in their interactions with macrophages during primary and secondary differentiation. The adherence signal for lymphocytes thus may be similar to that proposed for other blood cells.     

Plasma membrane lipid order of leukemic and normal immature avian erythroid cells

Mammalian erythroblasts and their leukemic counterparts contain characteristic disordered regions of plasma membrane identified as putative membrane protein collection sites. In order to determine whether erythroid cells which do not enucleate contain homologous membrane domains, immature avian erythroid precursor cells and avian erythroleukemic cells were examined using merocyanine 540 (MC540), a fluorescent dye whose binding is sensitive to the packing of membrane lipids. Results were found to contrast with previous studies of the murine equivalents of these cells. In birds, normal erythroid precursors, including basophilic erythroblasts from the bone marrow and spleen of anemic animals, contained no detectable (less than 0.1%) cells which were stained by the dye. But cells from chicks infected with avian erythroblastosis virus (AEV) did stain. Considering the pattern of staining observed on AEV-erythroblasts relative to other leukemic and normal phenotypes, however, we conclude that neither normal nor leukemic avian erythroid cells contain a functional equivalent to the membrane protein collection sites found on their mammalian counterparts.     

Phospholipid asymmetry in human erythrocyte ghosts

Using phospholipase digestion and the fluorescent probe merocyanine 540 the maintenance of phospholipid asymmetry in the plasma membrane of human erythrocyte ghosts was investigated. Digestion with phospholipase A2 indicated that ghosts prepared in the presence of Mg++ as the only divalent cation retained the normal phospholipid asymmetry characteristic of intact erythrocytes. These ghosts, like normal erythrocytes, also failed to stain with merocyanine 540. However, the presence of as little as 5-10 microM Ca++ during ghost preparation resulted in ghosts in which lipid asymmetry had been abolished, as indicated by phospholipase digestion. Moreover, these ghosts stained with merocyanine 540. In contrast to ghosts, intact erythrocytes treated with ionophore required millimolar levels of Ca++ ions to disrupt membrane lipid asymmetry. To discover the reason for this difference in behavior between ghosts and intact cells, ghosts were prepared from preswollen cells using only small volumes of buffer for lysis. These experiments demonstrated that as the cellular contents of erythrocytes are diluted, the asymmetric arrangement of phospholipids becomes more sensitive to disruption by Ca++.     

Insertion of lipid domains into plasma membranes by fusion with erythrocytes

After prelabeling the plasma membrane with several lipid-specific fluorescent probes, erythrocytes with symmetric lipid bilayers were fused with culture cells using either poly(ethylene glycol) or Sendai virus as fusogen. Several nonspecific probes were transferred to, and became uniformly distributed within, the culture cell membrane upon fusion. In contrast, when merocyanine 540, which displays preferential binding to bilayers in which the lipids are loosely packed, was used to prelabel erythrocytes, fluorescence remained localized within a small confined area of the membrane, even 24 h after fusion. These results suggest that insertion of the lipids of the erythrocyte membrane into the plasma membrane of the culture cell can produce discrete domains which persist as such for long periods following fusion. Because the inserted proteins of the erythrocyte membrane similarly do not freely diffuse throughout the culture cell membrane, interactions between membrane proteins and lipids may be involved in this singular compartmentalization.     

Distribution of concanavalin A in fibroblasts: direct endocytosis versus surface capping.

Indirect immunofluorescence of intact or acetone-extracted cells has allowed us to distinguish concanavalin A (Con A) which is associated with the plasma membrane of CHO cells from Con A which has been interiorized. We find that Con A is directly endocytized by these cells with no intervening stage of plasma membrane aggregation. The lectin accumulations observed by direct fluorescence are actually cytoplasmic collections of pinosomes which contain Con A. Only in a small fraction of CHO cells are true plasma membrane aggregates, or caps, found. This predominance of direct pinocytic interiorization over capping was not affected by dibutyryl cAMP or by treatments which can disrupt microtubules, including cold shock or exposure of the cells to anti-mitotic agents. Cytochalasin B, however, inhibited the uptake of Con A and at the same time promoted the formation of large surface aggregates of the lectin, or minicaps. Capping may reflect a competition between aggregation in the plane of the membrane and direct interiorization of bound lectin. Surface cap formation may be a characteristic process of cells with very low endocytic rates, such as lymphocytes.     

Cholesterol-free phospholipid domains may be the membrane feature selected by N epsilon-dansyl-L-lysine and merocyanine 540

We have used N epsilon-dansyl-L-lysine as a fluorescent membrane probe, to study cells taken from tissues concerned with immune function. There is a striking similarity between the staining selectivity of this compound and that reported by others for merocyanine 540. Both compounds stain leukemic, human, peripheral leukocytes, an erythroleukemia line, and some mouse bone marrow cells, suggesting common selectivity for a membrane feature of hemopoietic cells. Both compounds fail to stain red blood cells, normal human leukocytes, mouse spleen and thymus cells. We have recently reported that dansyl-lysine apparently selects for cholesterol-free phospholipid domains in liposomes and now report similar selectivity for merocyanine 540 staining of liposomes.     

Cooperative binding of concanavalin A to thymocytes at 4 degrees C and micro-redistribution of concanavalin A receptors.

The mode of binding of 125I-labelled concanavalin A and succinyl-concanavalin A to rat thymocytes at 4 degrees C was investigated. Simultaneously, the free binding sites of the cell-bound lectin molecules were quantified by horseradish peroxidase binding. Concanavalin A showed cooperative binding while succinyl-concanavalin A did not. The number of molecules of concanavalin A bound to the cell surface when it was saturated was twice the number of molecules of succinyl-concanavalin A. We interpret these results as showing that the binding of native concanavalin A to thymocytes at 4 degrees C brings about a cooperative modification of the membrane which leads to appearance of new receptors. Divalent succinyl-concanavalin A has no such effect. Horseradish peroxidase binding to cell-bound lectin was shown to be related to the immobilization of membrane receptors; the more they are immobilized, the more receptor-associated lectin can bind horseradish peroxidase. This allowed us to establish that post-binding events, which we called micro-redistribution, occurred at 4 degrees C when either concanavalin A or succinyl-concanavalin A binds to cells. A cooperative restriction of the micromobility of cell receptors is produced by increasing concentrations of concanavalin A. Succinyl-concanavalin A does not restrict cell receptor mobility at any concentration tested. The results are discussed in terms of cell stimulation and cell agglutination.     

Identification of concanavalin A receptors and galactose-binding proteins in purified plasma membranes of Dictyostelium discoideum

Two techniques have been modified to provide simple means for the identification of molecules which bind concanavalin A (Con A). Crossed immunoelectrophoresis was altered by replacing antibody with Con A, and receptors were identified by the precipitin arcs which they produced. Con A, tagged with fluorescein isothiocyanate, was also diffused into prefixed sodium dodecyl sulfate (SDS)-polyacrylamide gels, and additional receptors identified by fluorescence. More than 35 molecules in the plasma membranes of the cellular slime mold Dictyostelium discoideum which bind Con A were identified with these techniques. At least 12 of these diminish and 12 increase in importance as receptors during differentiation of the cells from the vegetative to the preculmination stage of development. In the course of these experiments, it was possible to confirm the presence of the galactose-binding protein discoidin, in the plasma membrane, by electrophoresing membrane proteins into an agarose gel. This lectin regains its sugar-binding activity after denaturation and electrophoresis in SDS.     

Molecular changes in the membranes of mouse erythroid cells accompanying differentiation.

The development of the mouse erythroblast to a mature erythrocyte is accompanied by changes in the composition and properties of the plasma membranes of these cells. Using double fluorescence techniques, we have simultaneously determined the distribution of lectin receptors and spectrin on the membranes of these cells. The lateral mobility of the lectin receptors in the membranes decreases as differentiation proceeds, and this is accompanied by an increasing concentration of spectrin associated with the membranes. The most significant concentration of spectrin occurs, however, during the enucleation of the late erythroblast, where we observe a complete segregation of the spectrin to the incipient reticulocyte, as well as a previously observed enrichment of receptors for concanavalin A into the plasma membrane surrounding the extruding nucleus. On the basis of these and other observations, we explore the possible molecular mechanisms involved in erythroblast enucleation and the role of spectrin in the regulation of protein mobility in erythroid cell membranes.     

An analysis of concanavalin A-mediated agglutination in two Chinese hamster ovary subclones whose surface phenotypes respond to maintenance in medium supplemented with dibutyryl cyclic AMP. V. Biochemical composition of the plasma membrane.

We have used two Chinese hamster ovary subclones whose surface phenotype has been extensively investigated with regard concanavalin A-mediated cell-cell agglutination and concanavalin A-induced receptor site clustering to investigate what changes in membrane composition, if any, can be correlated with the concanavalin A-detected changes in surface phenotype. These cell clones are uniquely disposed for this purpose since maintenance of the cells under different growth conditions produces changes in agglutinability and receptor site mobility in one cell clone (H-7W) but not the other (K-1). After extensive characterization of the surface membranes of these two subclones we have been unable to identify any change in the membrane peptides, glycopeptide, cholesterol, or fatty acid composition which can be directly correlated with the concanavalin A-detected surface phenotypes. It is of particular interest to note that we have been unable to correlate the presence or absence of the large external transformation-sensitive glycoprotein with the relative mobility of the lectin receptors or with the degree of concanavalin A-mediated cell agglutination. Furthermore we have been unable, in this system, to corroborate earlier data suggesting a role for cholesterol in determining the relative mobility of the lectin receptors. Thus using a cell system consisting of genetically matched cell clones, we have been unable to identify any changes in the biochemical composition of the plasma membrane which might be associated with the surface phenotypes detected by concanavalin A.     

Merocyanine 540 as a fluorescent probe of membranes: staining of electrically excitable cells.

With the exception of certain blood cells considered in the accompanying paper (Valinsky, Easton and Reich, 1978), merocyanine 540 (MC 540), a fluorescent membrane probe, selectively strains the membranes of a wide variety of electrically excitable cells, but not those of nonexcitable cells. This reaction is Ca2+-dependent when staining is performed in buffered iso-osmotic sucrose, Ca2+-independent when staining proceeds at high ionic strength, inhibited by La3+ and sodium Suramin, enhanced by controlled, low level photosensitization of cell-associated dye and essentially irreversible. These characteristics of the staining reaction depend upon the maintenance of both cell viability and a normal unperturbed membrane structure. Although the mechanisms involved in the staining specificity remain unknown, observation of MC 540 partitioning between benzene and water in model reactions indicates that dye transport into hydrophobic solvents is accompanied by the formation of stoichiometric complexes with cations and phospholipids. These results may suggest the existence of specific, possibly phospholipid-rich membrane domains that mediate complex formation with MC 540 in excitable cells; comparable domains either would not exist, or would be inaccessible at the external surfaces of nonexcitable cells.     

Involvement of spectrin in the maintenance of phase-state asymmetry in the erythrocyte membrane

The fluorescent probe merocyanine 540 does not stain the plasma membrane of normal human or murine erythrocytes, nor of genetically abnormal human spherocytic erythrocytes. It does, however, stain erythrocyte membranes in several systems in which the underlying spectrin network is altered or missing. Because of the greater affinity of merocyanine 540 for fluid—phase lipid bilayers, these results suggest that the external leaflet of erythrocyte membranes becomes more disordered upon alteration or loss of the internal spectrin network. Analysis of the transbilayer arrangement of membrane phospholipids by digestion with phospholipase A2 suggests that lipid compositional asymmetry of the erythrocyte membrane is responsible for a phase-state asymmetry between the two lipid leaflets, and that spectrin is required to maintain this asymmetry and the gel-like state of the external leaflet.     

Surface alterations in calf lymphocytes oxidized by sodium periodate.

In order to investigate alterations in surface structure in transformed lymphocytes, calf submandibular lymph node cell suspensions were oxidized with NaIO4. Oxidezed lymphocytes were morphologically transformed and had higher rates of DNA synthesis by 2 days after treatment. These results were prevented by reduction of the cell suspension with NaBH4, or by neuraminidase treatment of cells prior to oxidation. The amount of 125I-labeled Agaricus bisporus lectin bound to cells immediately after oxidation and the affinity constant for binding were increased over 2-fold, while cells immediately following oxidation and reduction showed decreased receptors with still higher affinity for the lectin compared to untreated cells. The amount of Phaseolus vulgaris lectin bound to oxidezed cells was also increased, but affinity was unchanged. Immediately following oxidation and reduction, these receptor sites were unchanged in number and affinity from untreated cells. In contrast, the number and affinity of receptors for concanavalin A were not changed immediately after oxidation or oxidation and reduction. In order to define the extent of compositional changes in surface glycoprotein receptors, plasma membranes were isolated from frozen calf submandibular lymph nodes. Compared to untreated plasma membranes, oxidezed membranes had similar contents of galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, fucose, and amino acids. Sialic acid content of oxidized membranes was reduced when measured by thiobarbituric acid assay. Sialic acids of untreated plasma membranes co-chromatographed with N-glycolylneurominic acid and N-acetylneuraminic acid, while those of oxidized membranes co-chromatographed with N-glycolylneuraminic acid and 5-acetamido-3,5-dideoxy-L-arabino-7-aldehydo-2-heptulosonic acid. Therefore, specific surface conformational changes in certain classes of membrane glycoproteins are associated with mild Malapradian oxidation of membrane sialic acids. These temporally precede NaIO4-induced transformation of calf lymphocytes. This is consistent with an hypothesis of membrane-mediated stimulation of lymphocyte transformation.