Differential lectin-mediated agglutinabilities of the embryonic and the first extraembryonic cell line of the early chick embryo

Summary The lectin-mediated agglutinability of cells dissociated from different areas of the gastrulating chick embryo was investigated. Differences in agglutinability were quantified by using a Coulter counter. Cells from the area pellucida (AP) and those from the endoderm of the area opaca (AOEn) are agglutinated by Concanavalin A (Con A), wheat germ agglutinin (WGA) andRicinus communis agglutinin (RCA). In cells from both areas the greatest agglutination response is obtained with RCA. Trypsinization of AOEn cells enhances their agglutinability with Con A, WGA and RCA. The lectin-induced agglutinability of cells from the area pellucida is similar in EDTA-dissociated and trypsinized cells.Cells from the AP are significantly more agglutinable with Con A than those of the AOEn regardless whether the former are obtained by trypsinization or dissociation with EDTA. The higher agglutinability of cells of the area pellucida with Con A, as well as the differential enhancement by trypsin of the agglutinability of AOEn cells with Con A, WGA, and RCA may reflect a difference in the cell surface glycoreceptors between the cells of the are pellucida (predominantly embryonic) and the first extraembryonic (AOEn) cell line. These cells have been shown to sort out from each other at the earliest stages of development.     

Agglunation of a transformed mouse cell line and a variant subline with concanavalin A: Effect of temperature and time of lectin incubation

Colchicine treatment enhanced Con A-mediated agglutination of erythrocytes to LM cells (LM is a “spontaneously” transformed mouse line) incubated for brief periods with Con A at 22° C. Longer incubations with Con A at 22° C rendered colchicine treated cells less agglutinable than untreated cells. Even short incubation times with Con A at higher temperature (37° C) rendered colchicine treated LM cells less agglutinable than their untreated counterparts. Below 15° C, colchicine treated cells remained more agglutinable than untreated cells even after long periods of Con A treatment. Cells of a variant clone (Rl) isolated from LM by negative selection with concanavalin A exhibited increased substratum adhesiveness and an absolute serum requirement. LM and variant cells exhibited a differential reponse to colchicine treatment, the variant subline reguiring longer periods of colchicine treatment to elicit changes in morphology and agglutinability.     

Classification of cell types: Agglutination and chromosomal properties

Summary The properties of agglutination by plant lectins, along with chromosome patterns, were examined in a variety of mammalian cell types. Untransformed adult and embryonic cells in culture, direct mouse spleen cell preparations, SV40-transformed 3T3 cells, and trypsinized 3T3 cells were all highly agglutinable with concanavalin A and with wheat germ agglutinin. In contrast, untreated cells of the contact-inhibited 3T3 line were alone among the cells tested in their low agglutinability. Chromosome analysis of the cultured cells showed that karyotypic variation from the diploid to an aneuploid state in mouse and rat embryo cultures was not accompanied by a change in agglutinability. Adult rat lung, adult monkey kidney, and embryonic human lung cells, which were all highly agglutinable, showed the normal diploid pattern. Thus, agglutination of cells by plant lectins appears to be a cellular property often associated with non-neo-plastic cells. This investigation was supported by Grants CA-12503 and ES-00260 from the National Institutes of Health, United States Public Health Service.     

Modulation of agglutinability by alteration of the surface topography in mouse ascites tumor cells

Factors involved in controlling agglutinability of cells with plant lectins include number, distribution, availability and mobility of cell surface lectin receptor sites. We have examined the concanavalin A (ConA)-mediated agglutination of mouse sarcoma 180 ascites tumor cells in the presence or absence of cytochalasin B (CB) using a quantitative electronic particle counter assay. These cells become substantially more agglutinable after brief treatment with low concentrations of CB. Scanning and transmission electron microscopy indicate that CB causes formation of large, broad, cell surface ruffles and loss of narrow projections that appear to be microvilli. Studies using fluorescent ConA suggest that lectin receptor sites concentrate on these ruffles and that the ruffles seem to directly mediate increased agglutinability in this system. Electron spin resonance studies suggest that CB does not alter lipid “fluidity” in these cells. The results indicate that the gross cell surface topography favoring high agglutinability is one displaying broad ruffles, not numerous narrow projections.     

Effect of metabolic state on agglutination of human erythrocytes by concanavalin

Intact freshly drawn or stored human erythrocytes, which show little agglutination by concanavalin A, become agglutinable by this lectin in the presence of adenosine. α-Methylglucose (10 mM) completely inhibits this agglutination. The concanavalin A agglutination shows no sensitivity to vinblastine or cytochalasin B.Resealed membranes prepared with ATP in lysing and resealing medium give modest agglutinability, while the presence of adenosine in both the lysing and the resealing medium results in a substantial agglutinability of the resealed membranes.Mild trypsin treatment of the erythrocytes causes an enhanced sensitivity to adenosine activation of the concanavalin A agglutination, while extensive trypsin treatment produced highly agglutinable erythrocytes that show no response to the presence of adenosine in the lectin solution. The extensively treated erythrocytes also show concanavalin A agglutination at temperatures below 37°C, under conditions in which intact or moderately treated erythrocytes do not agglutinate, with or without adenosine present.Results suggest that the adenosine activation of concanavalin A agglutination of intact human erythrocytes is mediated through a metabolic conversion of adenosine to a rapidly turned over metabolite which participates directly in the activation of agglutination. The agglutinability does not appear to depend on whole cell ATP levels, but may involve a particular pool of ATP.The effect of variation of cellular metabolic state and the response of particular systems involved in lectin-mediated agglutinability to cellular metabolism seem to be worth consideration in explaining the frequently large differences in agglutinability of und in cells indifferent biological states, such as those encountered in normal and transformed cells.     

Effect of metabolic state on agglutination of human erythrocytes by concanavalin A.

Intact freshly drawn or stored human erythrocytes, which show little agglutination by concanavalin A, become agglutinable by this lectin in the presence of adenosine. alpha-Methylglucose (10 mM) completely inhibits this agglutination. The concanavalin A agglutination shows no sensitivity to vinblastine or cytochalasin B. Resealed membranes preparaed with ATP in lysing and resealing medium give modest agglutinability, while the presence of adenosine in both the lysing and the resealing medium results in a substantial agglutinability of the resealed membranes. Mild trypsin treatment of the erythrocytes causes an enhanced sensitivity to adenosine activation of the concanavalin A agglutination, while extensive trypsin treatment produced highly agglutinable erythrocytes that shown no response to the presence of adenosine in the lectin solution. The extensively treated erythrocytes also show concanavalin A agglutination at temperatures below 37 degrees C, under conditions in which intact or moderately treated erythrocytes do not agglutinate, with or without adenosine present. Results suggest that the adenosine activation of concanavalin A agglutination of intact human erythrocytes is mediated through a metabolic conversion of adenosine to a rapidly turned over metabolite which participates directly in the activation of agglutination. The agglutinability does not appear to depend on whole cell ATP levels, but may involve a particular pool of ATP. The effect of variation of cellular metabolic state and the response of particular systems involved in lectin-mediated agglutinability to cellular metabolism seem to be worth consideration in explaining the frequently large differences in agglutinability of und in cells in different biological states, such as those encountered in normal and transformed cells.     

Studies on lectin-induced agglutination of Drosophila embryonic cell lines

The agglutination responses of three Drosophila cell lines to concanavalin A and wheat germ agglutinin have been examined. Although the cell lines were originally derived from late embryonic stages of the Ore-R strain of Drosophila melanogaster, they show quantitative differences in lectin-induced agglutination. Line 1 cells were least agglutinable with both lectins. All three cell lines reached maximum agglutination with concanavalin A concentrations at 25 μg/ml, but the agglutination response to wheat germ agglutinin was biphasic such that an initial rapid increase in agglutination with concentrations up to 25 μg/ml was followed by slower agglutination above this concentration. Cells of lines 1 and 2 from ten-day old cultures exhibited greater lectin-induced agglutination than cells from three-day old cultures. Age-dependent differences were not found for line 3 cells which gave maximum agglutination responses in both young and old cultures. Cell agglutination by concanavalin A was almost completely inhibited by pretreatment of the lectin with methyl-α-d-mannopyranoside, but preincubation of wheat germ agglutinin with N-acetyl-d-glucosamine caused only partial blockage. Lectin-induced agglutination was not reversible by treatment with the monosaccharide inhibitors. These observations have been discussed with reference to the origin of the three cell lines and their cell surface properties.     

Lectin Agglutinability of Mammary Tumours with Differing Metastatic Colonisation Potentials

A new fully quantitative method for assessment of lectin agglutinability has been used in this investigation to compare the surface composition of cells from tumours with high and low pulmonary colonisation potential. As in our previous work, we have used only primary (i.e., naturally-occurring) mammary tumours in mice. It was found that agglutinability with the lectins Concanavalin-A and Wheatgerm agglutinin bore no relationship to the pulmonary colonisation potential of the primary mammary tumour. However, cells from disaggregated secondary deposits of tumours which manifested high colonisation potential were consistently less agglutinable than the cells of the primary tumours from which they were derived.     

Lectin-mediated agglutination of erythrocyte ghost membranes following depletion of membrane protein and intramembrane particles

Profound digestion of unsealed human erythrocyte ghosts with high concentrations of Pronase results in a near complete loss of intramembrane particles while trypsin digestion is less effective. The small vesicles formed by proteolysis are agglutinable by soybean agglutinin (SBA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA), but not concanavalin A (ConA). Densitometer tracings of Pronase-treated vesicles analyzed on SDS-polyacrylamide gels demonstrated no detectable protein or glycoprotein migrating slower than the marking dye. The vesicles showed a loss of 90% Lowry positive material (the remainder may be non-protein chromogens), near depletion of sialyl residues, no significant change in lipid composition, and equal amounts of phospholipid phosphorus compared to an equal volume of ghosts. The lipid material extracted from Pronase-derived vesicles or intact ghosts inhibited hemagglutination with SBA and WGA but not ConA. SBA but not ConA was found to specifically bind to Pronase-derived vesicles while both lectins bound to native ghosts. These observations suggest that neither the integrity of the intramembrane particles nor the presence of membrane glycoprotein appears essential for SBA-, WGA-, and PHA-mediated agglutination. Furthermore, it appears that native membrane glycolipids (and perhaps glycopeptides) can bind SBA, WGA and PHA. The membrane glycolipids may play a larger role than heretofore realized in lectin-mediated agglutination of cells.     

Quantitative agglutination of specific populations of sea urchin embryo cells with concanavalin A

It has been demonstrated that specific changes in carbohydrate-containing cell surface lectin receptor sites occur with differentiation and maturation of sea urchin embryo cells. In this study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicates that concanavalin A (Con A) mediated agglutination of dissociated 32/64 cell sea urchin embryos differs dramatically with respect to specific cell populations. The migratory cell type, the micromere, is significantly more agglutinable with Con A than the other cell types and colchicine treatment markedly increases sea urchin embryo cell agglutinability. The results indicate that like many malignant cells which display extensive migratory behavior, specific migratory populations of embryonic cells are agglutinable with Con A. The results are discussed with respect to the possible nature of lectin receptor sites on specific populations of embryonic cells and the possible role of colchicine-sensitive structures in controlling the display patterns of these sites.     

Agglutination of Sindbis Virus and of Cells Infected with Sindbis Virus by Plant Lectins

We have examined the agglutination of Sindbis virus and of chick and hamster cells infected with Sindbis virus by two of the plant lectins, concanavalin A and Ricinus communis agglutinin. Both lectins agglutinate the virus by binding to the polysaccharide chains of the envelope glycoproteins. Both chick and hamster cells exhibit increased agglutination by the lectins after infection by Sindbis virus. In the case of chick cells infected with Sindbis virus, this increase in agglutinability occurs between 3 and 5 h after infection. Infected and mock-infected cells bind the same amount of (3)H-labeled concanavalin A, which suggests that the increase in agglutination after infection is due to rearrangements at the cell surface rather than to insertion of new lectin binding sites per se.     

Lectin-binding properties of hamster egg zona pellucida and plasma membrane during maturation and preimplantation development

The structure and organization of the zona pellucida and plasma membrane of the hamster egg at various stages of maturation and development were examined using lectin-mediated agglutination and the binding of fluorescent-labeled lectins. Ricinus communis I and Dolichos biflorus lectins specifically agglutinated the zona pellucida of both unfertilized and fertilized eggs, while wheat germ agglutinin (WGA) only agglutinated eggs which had been pretreated with protease. Six other lectins failed to agglutinate even eggs pretreated with protease. A comparison of the lectin-binding sites on the zona pellucida of eggs in various stages of maturation and development revealed that the intensity of binding and distribution of fluorescent-labeled lectins remain unchanged. Zona-free eggs were agglutinated by every lectin tested except those recognizing -fucose-like residues. Fertilized zona-free eggs were slightly more agglutinable by concanavalin A (ConA), Lens culinaris and WGA than unfertilized eggs. When the surfaces of zona-free eggs were examined with fluorescent ConA, Ricinus communis I and WGA, maximal binding was seen when eggs reached full maturity and binding decreased during the later stages of preimplantation development.     

Polyadenylation of nascent RNA during the embryogenesis of Ilyanassa obsoleta.

It has been demonstrated that specific changes in carbohydrate-containing cell surface lectin receptor sites occur with differentiation and maturation of sea urchin embryo cells. In this study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicates that concanavalin A (Con A) mediated agglutination of dissociated $\text{32}\text{64}$ cell sea urchin embryos differs dramatically with respect to specific cell populations. The migratory cell type, the micromere, is significantly more agglutinable with Con A than the other cell types and colchicine treatment markedly increases sea urchin embryo cell agglutinability. The results indicate that like many malignant cells which display extensive migratory behavior, specific migratory populations of embryonic cells are agglutinable with Con A. The results are discussed with respect to the possible nature of lectin receptor sites on specific populations of embryonic cells and the possible role of colchicine-sensitive structures in controlling the display patterns of these sites.     

Surface alterations in transformed epithelial and fibroblastic cells in culture: A disturbance of membrane degradation versus biosynthesis?

Fibroblasts which were transformed with oncogenic DNA or RNA viruses as well as by chemicals or spontaneously have, with very few exceptions, been shown to possess increased agglutinabilities with various plant agglutinins. This surface membrane alteration is now shown to hold true also for transformed epithelial cells and for tumors of epithelial origin as monitored by wheat germ agglutinin, Concanavalin A, phytohemagglutinin, Ricinus communis and Great Northern bean (GNB) agglutinin. An agglutinin from lentils did the opposite. It permitted the agglutination of normal liver cells but not that of hepatoma cells which led to the suggestion that some membrane glycoproteins or glycolipids may possibly be turned inside out while others are turned outside in (during the transformation process. Inhibition of protein synthesis in normal cells brought about the agglutinable membrane state in non-growing or resting cells but not in actively growing cells. Since this agglutinability could be inhibited by protease inhibitors and since proteolytic enzymes could bring about the agglutinable membrane state in growing normal cells, it was suggested that growing cells have as active a biosynthesis of membrane proteins as do non growing cells but that non-growing cells are actively catabolizing their membranes with proteases, much more so than non growing, stationary cells.     

Cell surface properties of L1210 leukaemia cells.

The surface properties of vincristine-colchicine sensitive and resistant L1210 leukaemic cells have been studied using concanavalin A mediated agglutination assay as well as electron microscopic visualization of concanavalin A receptors. 3H-colchicine uptake by the sensitive and resistant lines has also been compared. The resistant L1210 leukaemic cells proved less agglutinable than the sensitive ones at the same concanavalin A concentration. Previous treatments with either colchicine, vincristine or chlorpromazine caused a marked decrease in the agglutinability of the sensitive L1210 leukaemic cells, while agglutination of the resistant ones was lowered slightly by the same treatments. The 3H-colchicine uptake of the sensitive cells was three times higher than that of the resistant ones.     

Glutaraldehyde fixation and the mechanism of erythrocyte agglutination by concanavalin a and soybean agglutinin

To evaluate the importance of lectin receptor mobility and clustering for enhanced cell agglutinability, the effect of glutaraldehyde fixation on the agglutinability of human erythrocytes by concanavalin A and soybean agglutinin was investigated. Agglutinability was evaluated in unperturbed Microtiter^® plates. Fixation increased slightly the agglutinability of the erythrocytes by both lectins. Fixation did not alter trypsin-enhanced agglutinability. Furthermore, when fixed erythrocytes were trypsinized, their agglutinability increased to the level of unfixed, trypsinized erythrocytes.The kinetics of agglutination of fixed and unfixed erythrocytes were monitored in an electronic particle counter. The shear forces associated with the kinetic experiments diminished fixed-cell to fixed-cell agglutination, i.e., both lectins gave slower kinetics of agglutination with fixed erythrocytes than with unfixed erythrocytes. In contrast, the kinetics of concanavalin A-mediated agglutination of trypsinized-fixed erythrocytes mixed with equal numbers of trypsinized-unfixed erythrocytes were indistinguishable from the rapid kinetics of agglutination of trypsinizedunfixed erythrocytes alone. Light microscopy revealed aggregates composed of fixed and unfixed erythrocytes.We conclude that glutaraldehyde fixation does not diminish the agglutinability of human erythrocytes under low-shear conditions. Our results indicate that the enhanced agglutination of trypsinized erythrocytes is not dependent on clustering of lectin receptors. The disruption of agglutination of fixed erythrocytes by shear forces that do not disrupt agglutination of fixed erythrocytes with unfixed erythrocytes suggests that the rigidity of the fixed erythrocyte may prevent stable aggregate formation by fixed erythrocytes alone.     

Binding of I-Concanavalin a and agglutination of embryonic neural retina cells : Age-dependent and experimental changes

Embryonic chick neural retina cells dissociated from retina tissue by treatment with EGTA (a calcium chelator) show an age-dependent decline in ability to agglutinate with concanavalin A (ConA). This developmental change in cell surface properties is not due to loss of ConA-binding sites, since mature retina cells can be rendered agglutinable by mild trypsinization. It is also not due to masking of ConA receptors, or to a decrease in their amount, since retina cells from late embryos (19 days) bind four times as much ¹²⁵I-ConA as cells from early embryos (8 days). Our findings lead us to suggest that, as the retina differentiates the lateral mobility of ConA receptors in the cell membrane decreases resulting in a reduction of cell agglutinability; trypsinization of late embryo retina cells increases the mobility of the receptors and thereby facilitates their clustering by the lectin into a configuration conducive to cell agglutination.The ability of late embryo (19 day) retina cells dispersed with EGTA to agglutinate with ConA could be increased by still other treatments: by pre-incubation of the cell suspension in Tyrode's balanced salt solution (1 h, 37 °C); and by brief pre-exposure to glutaraldehyde. These two treatments did not enhance cell agglutination with wheat germ agglutinin (WGA). Glutaraldehyde treatment of trypsinized cells made them agglutinable with ConA also at 4 °C; cells treated otherwise agglutinated only at higher temperature. Surface-saturation of monodispersed retina cells with ConA at 37 °C—but not at 4 °C—prevented their agglutination with this lectin, but not with WGA; this inhibition was reversible by methyl a-D-glucopyranoside (αMG).     

A quantitative assay for concanavalin A- and Ricinus communis agglutinin-mediated agglutinations of rat ascites hepatoma cells: Relationship between concanavalin a binding and cell agglutination

A simple quantitative assay method was developed for the agglutination of rat ascites hepatoma cells mediated by Concanavalin A or Ricinus communis agglutinin. This method was based on the principle that the turbidity of a cell suspension is proportional to the sum of the cross-sectional area of cells and aggregates. As predicted by the theoretical consideration, the turbidity decreased when cells were aggregated and the decrease was a function of the average number of the cells in aggregates.The agglutinability of the cells, judged by this method, showed a maximum value at a certain concentration of the agglutinin. By further addition of the agglutinin, the agglutinability slightly decreased from the maximum. These phenomena were observed both for Concanavalin A and Ricinus communis agglutinin. The binding and the agglutination experiments using [³H] concanavalin A revealed that the binding to approx. 20% of the total receptors caused a maximal agglutination. This suggested that the receptors responsible for the agglutination constitute only a small part of the total receptors on the surface.     

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.     

Fluorescein Isothiocyanate-Labeled Lectin Analysis of the Surface of the Nitrogen-Fixing Bacterium Azospirillum brasilense by Flow Cytometry

The cell surface of Azospirillum brasilense was probed by using fluorescein isothiocyanate (FITC)-labeled lectins, with binding determined by fluorescence-activated flow cytometry. Cells from nitrogen-fixing or ammonium-assimilating cultures reacted similarly to FITC-labeled lectins, with lectin binding in the following order: Griffonia simplicifolia II agglutinin > Griffonia simplicifolia I agglutinin > Triticum vulgaris agglutinin > Glycine max agglutinin > Canavalia ensiformis agglutinin > Limax flavus agglutinin > Lotus tetragonolobus agglutinin. The fluorescence intensity of cells labeled with FITC-labeled G. simplicifolia I, C. ensiformis, T. vulgaris, and G. max agglutinins was influenced by lectin concentration. Flow cytometry measurements of lectin binding to cells was consistent with measurements of agglutination resulting from lectin-cell interaction. Capsules surrounding nitrogen-fixing and ammonium-assimilating cells were readily demonstrated by light and transmission electron microscopies.