Co-operativity of lectin binding to fibroblasts and its relation to cellular actomyosin

1979 ◽  
Vol 39 (1) ◽  
pp. 117-136
Author(s):  
D. Thom ◽  
D.S. Cox ◽  
R. Safford ◽  
D.A. Rees
Keyword(s):  
Concanavalin A ◽  
Cellular Response ◽  
Cytochalasin B ◽  
Shape Change ◽  
Lectin Binding ◽  
Reciprocal Relationship ◽  
Hill Coefficient ◽  
Cell Detachment ◽  
Con A ◽  
Surface Receptors

We have investigated the binding of 125I-concanavalin A (125I-Con A) and 125I-succinyl concanavalin A (125I-s-Con A) to rat fibroblasts (16C line) as a function of the concentration of added lectin, and the alterations to this binding behaviour caused by drugs which modify the cytoskeleton. The changes in cell behaviour which occur at different levels of binding have also been studied. As shown previously for some other systems, the binding of Con A is complex and partly co-operative. Three phases can be distinguished in our system: (i) pre-nucleation binding, (ii) binding which shows a small positive slope in a Scatchard plot and a Hill coefficient greater than unity, and which therefore is incipiently co-operative, and (iii) post-co-operative binding. The co-operative phase of binding is paralleled by progressive inhibition of EGTA-mediated cell detachment from substrata, with inhibition being complete when this phase of binding is complete. Likewise, the phagocytosis of latex spheres is progressively inhibited up to a threshold which coincides with the completion of co-operative binding. Thirdly, cells pretreated with Con A round up with colchicine (10(−5) M) if co-operative finding is complete, but adopt broad epithelial shapes if it is not. s-Con A does not show cooperative binding, and correspondingly does not inhibit EGTA-mediated cell detachment, or show a distinct threshold in the inhibition of phagocytosis, or promote the 2 types of shape change with colchicine. The pattern of Con A binding is drastically altered by pretreatment of cells with cytochalasin B or azide. The Scatchard and Hill plots show that the co-operative phase remains and is complete at about the same level of binding, but that it is more readily nucleated and takes place against a changed number and/or distribution of receptors. Pretreatment of cells with colchicine causes changes in the pattern of binding which are different from those observed with cytochalasin B or azide and are more difficult to interpret. We conclude that a reciprocal relationship exists between the cellular actomyosin and the state of cell surface receptors. Perturbation of actomyosin by cytochalasin B or azide can enhance the freedom of some receptors to participate in a co-operative rearrangement which facilitates the binding of further molecules of lectin. Vice versa, the co-operative event has a feedback influence on the cellular actomyosin to cause alterations of cellular response.

scholarly journals Inhibition of intercellular adhesion by concanavalin A is associated with concanavalin A-mediated redistribution of surface receptors.

1979 ◽  
Vol 80 (1) ◽  
pp. 128-140 ◽  
Author(s):  
P C Letourneau
Keyword(s):  
Concanavalin A ◽  
Cytochalasin B ◽  
Atp Synthesis ◽  
Intercellular Adhesion ◽  
Temperature Sensitive ◽  
Con A ◽  
Retinal Cells ◽  
Surface Receptors ◽  
Receptor Complexes ◽  
Dose Dependent

The inhibition of adhesion between aggregates and layers of embryonic retinal cells by concanavalin A (Con A) and Con A-mediated rearrangements of Con A receptors on retinal cells were studied. A short incubation of aggregates and layers with 10 micrograms/ml Con A substantially reduced aggregate-to-layer adhesion in a subsequent assay without soluble lectin present. This effect of Con A was dose-dependent, temperature-sensitive, involved events subsequent to Con A binding, and was reduced by cytochalasin B. The inhibition produced by succinylated Con A was substantially increased by incubation with antibody to Con A. Visualization of ConA- receptor complexes by fluorescence microscopy revealed that binding of Con A induced clearing of Con A receptors from filopodia, flattened regions of growth cones, and the edges of axons. This clearing reaction was prevented by the same agents that reduced Con A's inhibition of cell adhesion: low temperature, succinylation of Con A, or cytochalasin B. Aggregate-layer adhesion was restored by releasing Con A at 37 degrees C. Inhibitors of protein and ATP synthesis did not prevent recovery of ability to make adhesions. However, release of Con A at lowered temperatures did not prevent recovery. The results suggest that intercellular adhesion is inhibited by events associated with redistribution of Con A-receptor complexes on retinal cells.

scholarly journals Antagonism by dibutyryl adenosine cyclic 3',5'-monophosphate and testololactone of concanavalin A capping.

1975 ◽  
Vol 66 (2) ◽  
pp. 392-403 ◽  
Author(s):  
B Storrie
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Cytochalasin B ◽  
Cold Treatment ◽  
Intracellular Camp ◽  
Camp Concentration ◽  
Con A ◽  
Cell Rounding ◽  
Intracellular Camp Concentration

Exposure of CHO-K1 cells in vitro to dibutyryl adenosine cyclic 3',5'-monophosphate (DBcAMP) plus testololactone produces a rapid, reversible antagonism of ligand-induced collection of initially dispersed concanavalin A (Con A) binding sites into a caplike mass. Morphologically, as Con A capping occurs, the cells become less spread and then round completely. With prolonged Con A exposure, cells cultured in either the absence or the presence of DBcAMP plus testololactone cap and round. Capping is blocked by cold treatment and respiratory inhibitors. Colcemid at concentrations greater than 1 muM promotes both Con A capping and cell rounding. Cytochalasin B at similar concentrations inhibits both capping and cell rounding. Treatment of cells with Con A has little effect on intracellular cAMP concentration. Possible mechanisms by which cAMP may modulate the movement of Con A binding sites are discussed.

scholarly journals Distribution of concanavalin A binding sites on the surface of dissociated rat submandibular gland acinar cells.

1975 ◽  
Vol 23 (8) ◽  
pp. 607-617 ◽  
Author(s):  
T Amakawa ◽  
T Barka
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Concanavalin A ◽  
Binding Sites ◽  
Lectin Binding ◽  
Single Cells ◽  
Apical Cell ◽  
Acinar Cells ◽  
Con A ◽  
Dissociated Cells

The submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation of divalent cations and mechanical force. A suspension of single cells was obtained in low yield by centrifugation in a Ficoll-containing medium. Immediately after dissociation and after a culture period of 16-18 hr the dissociated cells were tested for agglutinability by concanavalin A (Con A). Using ferritin (tfer)-conjugated Con A the lectin binding by the isolated acinar cells was also studied. The dissociated cells were agglutinated by low concentrations of Con A and bound Fer-Con A molecules on their entire surface without any indication of polarization of the cell membrane. There was a considerable cell to cell variation in the amount of Fer-Con A binding which was, in general, sparse and patchy. The contact surfaces between agglutinated cells revealed a dense binding of Fer-Con A molecules irrespective of the types of cells participating in the agglutination reaction. Cells cultured for 16-18 hr were no longer agglutinated by Con A. As compared to the freshly dissociated cells the cultured acinar cells revealed a more uniform and denser binding of Fer-Con A molecules. Furthermore, there were more lectin molecules bound to the cell surface corresponding to the basal part of the cell, where the nucleus and most of the rough surface endoplasmic reticulum were located, than to the apical cell surface. It is suggested that the higher density of lectin-binding sites on the cell surface in the vicinity of the cisternae of the rough endoplasmic reticulum indicates insertion sites of newly synthesized membrane glycoproteins.

scholarly journals Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

1977 ◽  
Vol 74 (3) ◽  
pp. 950-962 ◽  
Author(s):  
GL Nicolson ◽  
N Usui ◽  
R Yanagimachi ◽  
H Yanagimachi ◽  
Smith JR
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Similar Degree ◽  
Con A ◽  
Lectin Receptors ◽  
Surface Receptors ◽  
Epididymal Spermatozoa ◽  
Lectin Binding Sites

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.

scholarly journals Schistosomula of Schistosoma mansoni clear concanavalin A from their surface by sloughing.

1982 ◽  
Vol 94 (2) ◽  
pp. 355-362 ◽  
Author(s):  
J C Samuelson ◽  
J P Caulfield ◽  
J R David
Keyword(s):  
Schistosoma Mansoni ◽  
Concanavalin A ◽  
Binding Sites ◽  
Culture Medium ◽  
Culture Media ◽  
Lectin Binding ◽  
Defined Medium ◽  
Con A ◽  
Sds Page ◽  
Lectin Binding Sites

The lectin concanavalin A (Con A) was used as a model probe to study the behavior of molecules bound to the surface of recently transformed schistosomula of Schistosoma mansoni. Con A binding was saturable (150-180 pg/organism) and specifically competed by alpha-methyl mannoside. Both FITC-Con A and 125-I-Con A were lost from the surface of schistosomula with a halftime of 8-10 h in culture in defined medium. A comparable decrease in the binding of Con A to schistosomula cultured and then labeled with the lectin indicated that the labeling procedure itself was not inducing the observed change. Internalization of Con A was not seen by either fluorescence microscopy or electron microscope radioautography. In addition, 70-80% of the radioactivity lost from the parasite was recoverable by TCA precipitation from the culture medium as intact Con A (27,000 mol wt on SDS PAGE). Thus, the mechanism of clearance of bound Con A from the surface of cultured schistosomula is apparently by sloughing of Con A molecules intact into the culture media and not by endocytosis and degradation. Con A binding sites, visualized with hemocyanin by scanning electron microscopy, appeared homogeneously distributed over the surface of schistosomula when organisms were labeled at 4 degree C or after fixation with glutaraldehyde. However, Con A and hemocyanin formed aggregates on the surface of schistosomula when labeling was performed at 37 degrees C, which suggests that lectin binding sites have lateral mobility within the plane of the membrane. These aggregates are likely independent of metabolism by the parasite because aggregation also occurs on the surface of organisms killed with azide.

scholarly journals Surface properties related to concanavalin A-induced agglutination. A comparative study of several Entamoeba strains.

1977 ◽  
Vol 145 (3) ◽  
pp. 652-665 ◽  
Author(s):  
D Trissl ◽  
A Martínez-Palomo ◽  
C Argüello ◽  
M de la Torre ◽  
R de la Hoz
Keyword(s):  
Surface Properties ◽  
Concanavalin A ◽  
Room Temperature ◽  
Lectin Binding ◽  
Cationized Ferritin ◽  
Con A ◽  
Low Concentrations ◽  
Negative Surface ◽  
Negative Surface Charge ◽  
Axenic Strains

Pathogenic strains of Entamoeba histolytica are more easily agglutinated with concanavalin A (Con A) than strains isolated from human asymptomatic carriers. All three pathogenic strains studied here were found to agglutinate with low concentrations of Con A in contrast to various nonpathogenic axenic strains of amebas, characterized by their ability to grow at room temperature. Our present observations suggest that the extreme susceptibility of pathogenic strains of E. histolytica to agglutinate with Con A is related to their higher capacity for lectin binding and to their lack of detectable repulsive charges at the cell surface. The amount of fluorescein-tagged Con A bound to the surface was much higher in pathogenic strains. Only nonpathogenic strains showed a detectable negative surface charge as studied both by means of cell microelectrophoresis and by labeling cells with cationized ferritin at 0 degrees C. The mobility of surface Con A receptors estimated as the percentage of caps was comparable in all strains. Results of one strain cultured in axenic and monoxenic conditions suggested that bacteria can modify the behaviour of E. histolytica trophozoites by altering surface properties of the amebas.

Lectin Binding Demonstrates Different Glycoprotein Abnormalities In Thrombasthenic And Bernard-Soulier Platelets

1981 ◽  
Author(s):  
Philip A Judson ◽  
David J Anstee
Keyword(s):  
Arachis Hypogaea ◽  
Concanavalin A ◽  
Marked Reduction ◽  
Human Platelet ◽  
Lectin Binding ◽  
Minor Components ◽  
Polyacrylamide Gels ◽  
Con A ◽  
Platelet Membranes ◽  
Bernard Soulier Syndrome

We have previously shown that the lectins from Maclura aurantiaca (Ma) and Arachis hypogaea (Ah) (peanut) bind selectively to GP-I in human platelet membranes. In contrast Concanavalin A lectin binds primarily to GP-III. GP-I is deficient or absent from the membranes of individuals with Bernard-Soulier Syndrome (B-S.S.) while GP-II and III are deficient or absent from Thrombasthenic (G.T.) platelets. We have investigated the binding of radioiodinated lectins to SDS polyacrylamide gels of whole platelets from patients with B-S.S. and G.T. The object of this study was to define further the nature of the glycoprotein abnormalities in these conditions.The results clearly demonstrated a marked reduction in the binding of Ma and Ah lectins to the GP-I region of B-S platelets when compared to that of normal platelets. No new lectin binding components were observed. The binding of Con A lectins to B-S platelets did not appear to be significantly different from that to normal platelets. In contrast Ma and Ah binding to G.T. platelets appeared normal whereas the binding of Con A to GP-III was markedly reduced. Two faint Con A binding bands were apparent in the GP-III region of the G.T. platelets. It is not clear whether either of these represent residual GP-III or if they are minor components masked by GP-III in normal platelets. The carbohydrate of GP-I in normal platelets consists almost entirely of O-glycosidically linked oligosaccharides. Ah and Ma lectins bind selectively to galactosyl and N-acetylgalactosaminyl residues in this type of oligosaccharide. We conclude that B-S platelets have a gross deficiency of O-glycosidically linked oligosaccharides. Con A binds selectively to branched N-glycosidically linked oligosaccharides with a mannose-rich core. We conclude that such structures normally present on GP-III are grossly deficient in G.T. platelets.

The relationship between endocytosis of concanavalin A and phytohaemagglutinin receptors and blast transformation, and direct identification of individual rabbit lymphocytes reactive to both mitogens

1982 ◽  
Vol 56 (1) ◽  
pp. 141-156
Author(s):  
C. Raffel ◽  
S. Sell
Keyword(s):  
Cell Surface ◽  
Concanavalin A ◽  
Cell Activation ◽  
Lectin Binding ◽  
Lymphocyte Activation ◽  
Early Event ◽  
Blast Transformation ◽  
Lectin Receptors ◽  
Surface Receptors ◽  
Rabbit Lymphocytes

Distribution and modulation of rabbit lymphocyte phytohaemagglutinin acceptors and concanavalin A acceptors during activation of rabbit lymphocytes have been examined by electron microscopy. Two types of cell surface acceptors have been tentatively identified, lectin binding acceptors that do not modulate, and receptors that are endocytosed when blast transformation is stimulated. All of the cells have binding acceptors for both lectins. Endocytosis correlates with early blast transformation and serves as an early marker for lymphocyte activation. When examined after 24 h of culture, those cells that undergo blast transformation contain endocytosed lectin receptors, whereas small untransformed cells do not. Capping prior to endocytosis is rarely observed. The mechanism whereby the signal for transformation is maintained after the reaction of lectin with cell surface receptors and transposed to the nucleus is not known. Although we conclude that endocytosis is an early event required for cell activation, it is possible that endocytosis is secondary to other activation events. By evaluation of sequential endocytosis, individual rabbit lymphocytes that endocytose only concanavalin A, only phytohaemagglutinin, both concanavalin A and phytohaemagglutinin, or neither lectin, have been identified.

Cell Agglutination Mediated by Concanavalin A and the Dynamic State of the Cell Surface

1974 ◽  
Vol 14 (1) ◽  
pp. 197-202
Author(s):  
M. INOUE
Keyword(s):  
Cell Surface ◽  
Concanavalin A ◽  
Binding Sites ◽  
Dynamic Condition ◽  
Dynamic State ◽  
Con A ◽  
Cell Agglutination ◽  
Surface Receptors ◽  
Topographical Distribution ◽  
Ehrlich Ascites Tumour

The binding of 131I-labelled concanavalin A (131I-Con A) to the cell surface has been studied in Ehrlich ascites tumour cells (EATC) and beef erythrocytes under various conditions. The binding of concanavalin A (Con A) to the cell surface was very specific and the available binding sites were saturated within a few minutes. The amount of 131I-Con A bound to EATC was 4.14 x 107 molecules/cell at 37 °C and 2.12 x 107 molecules/cell at 0 °C. Under these conditions, cell agglutination was observed only at 37 °C and not at 0 °C. However, the binding sites measured at 0 °C were also effective for agglutination at 37 °C. Beef erythrocytes were agglutinated by Con A only after treatment of cells with papain. The number of binding sites for Con A on the cell surface was decreased by this treatment to about half the number present on untreated cells. Various reagents such as colchicine, monoiodoacetic acid, dinitrophenol, rotenone, sodium azide and carboxyl cyanide-m-fluorophenylhydrazone (FCCP) had no effect on Con A-mediated cell agglutination. In contrast, periodate treatment produced a remarkable decrease in the agglutinability of cells. From these data, it is concluded that the cell agglutination induced by Con A was due to the topographical distribution of the surface receptors for the lectin, and not the result of energy-dependent or microtubule-dependent reaction processes. The number and the state of Con A receptors on the cell surface were in a dynamic condition, their conformation, orientation, and/or topographical distribution changing under different conditions.

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