Cell surface changes during the differentiation of Dictyostelium discoideum

Printed in Sweden Copyright 8 1975 by Academic Press, Inc. All rights of veproduction in any fovmIESEII~/

Experimental Cell Research 92 (1975) 372-382

CELL SURFACE CHANGES DURING OF DICTYOSTELIUM Interaction

THE DIFFERENTIATION DISCOIDEUM

of Cells with Concanavalin

A

CLAIRE WEEKS and G. WEEKS Department of Microbiology, University of British Columbia, Vancouver, B.C. V6T IW5 Canada

SUMMARY The parameters affecting the agglutination of cells of Dictyostelium discoideum by Concanavalin A (ConAl have been investigated. Under the incubation conditions emoloved. incubation time does not markedly affect agglutination, but there are distinct optima for cell density and gyration speed. Agglutination does not occur at low temperatures, but the transition temperature between the unagglutinated and fully agglutinated states is markedly influenced by ConA concentration. The rate of aggregation of strain NC4 is considerably reduced by Con A. In contrast, the differentiation of strain Ax-2 in the presence of ConA is either unaffected or only slightly inhibited, depending on the incubation conditions. Succinylated-ConA binds to the same sites as the unmodified lectin, but has no effect on the differentiation of strain NC4, suggesting that ConA binding sites are not directly involved in cellcell contacts vital to the differentiation of D. discoideum. There is a gradual decrease in the susceptibility of cells of D. discoideum to agglutination by Con A as the cells pass from exponential growth phase.to stationary growth phase in axenic medium and from vegetative amoebae to aggregates on a solid substratum. These results provide quantitative evidence for a gradual change in carbohydrate containing binding sites during differentiation.

It has now been conclusively demonstrated that malignant cells are far more susceptible to agglutination by the plant lectin Concanavalin A (ConA) than are their normal counterparts [l]. This phenomenon is believed to reflect differences in cell surface architecture, although the precise nature of the difference has not been definitively established. More recently Con A has been used to monitor changes in surface structure of cells in a number of differentiating systems: chick neural retina cells [2, 31, human intestinal cells [4]; Dictyostelium discoideum [5], and seaurchin embryo cells [6]. In all these studies Exptl Cell Res 92 (1975)

a common pattern has emerged; as cells become more differentiated they become less susceptible to agglutination by Con A. The present report describesthe parameters affecting the agglutination of D. discoideum by Con A and the time dependency of the surface changes during differentiation, as measured by altered susceptibility to agglutination by this lectin. We have also studied the effects of ConA on differentiation, in order to obtain information on the possible role of cell surface Con A binding sites in the differentiation process.

Cell surface changes during differentiation

METHODS Organisms and culture conditions Dictyostelium discoideum strains NC-4 and Ax-2 were obtained from Dr J. M. Ashworth. Strain NC-4 was grown in association with Aerobacter aerogenes on nutrient agar plates as described previously [7]. Strain Ax-2 was grown in complex media [5], except that the content of Difco yeast extract had to be doubled (see table 2) to maintain the previously reported growth rates [8]. This modification of the growth media did not quantitatively affect any of the agglutination assays reported previously [5]. Both strains were harvested by-centrifugation at 700 g, and washed with distilled water. The cell viability was determined by counting colony forming units after growth in association with Aerobacter aerogenes [5].

Materials ConA (3 x crystallized) was obtained from MilesYaeda as a suspension in saturated ammonium sulphate. For experiments conducted at low concentrations of the lectin, ConA was diluted with saturated NaCl, while for experiments at high concentrations, Con A was dialysed against saturated sodium chloride for 2 h at 4°C. Succinylated (Succ-ConA) ConA was prepared as described previously [9] and purified by chromatography on G-100 Sephadex.

Agglutination

assays

Unless otherwise indicated. washed cells were resuspended in buffered Banner’s salts solution (KCl, 1 x 10-e M: NaCl. 1 x 1O-z M: CaCl,. 1.5 x lo-* M: Tris-Cl, pH 7.5, 1’~ 1O-s M) (10). NaCl (110 pmolesj and varying amounts of ConA were added to 5 ml of cell suspension, and the suspension was then incubated at 22°C in a 25 ml Erlenmeyer flask on a gyratory shaker at 200 rpm. After 10 min incubation, agglutination was quantitated microscopically by counting the unagglutinated cells [5]. In some experiments ConA dependent agglutination was plotted against ConA concentration in order to determine the Con A concentration that was necessary to produce 50 % agglutination.

313

and VtiriOUs concentrations of Con A (SO-800 ,ug/ml) for 10 min at 22°C in a gyratory shaker. Aliquots of cell susnension (0.3 ml) were then dispensed onto WhatmanNo. 50 filter papers and incubated at 22°C. (b) Washed cells were resuspended in buffered Banner’s salts solution at a cell density of 5 x 10’ cells/ml and incubated in the presence of 110 pmoles/ ml NaCl and various concentrations of Con A (50-8OO,ug/ml) for 10 min at 22°C in a gyratory shaker. Aliquots (0.2 ml) were spread onto the surface of 2 % agar in 60 mm Petri plates. The Petri plates were incubated at 22°C to allow differentiation to proceed. (c) In some exoeriments. washed cells were resuspended in buffered Bonner’s salts solution at 5 x 10’ cells/ml and then dispensed directly onto 2 % agar plates containing 110 pmoles/ml NaCl and various concentrations of Con A (S-800 pg/ml) dissolved in the agar. (d) Washed cells were resuspended in buffered Bonner’s salts solution at 2.5 x lo6 cells/ml and 0.5 ml aliquots were incubated in the presence of 22 pmoles/ ml NaCl and 1.6160 pg/ml ConA, in Van Tiegham cells at 22°C 1101.The Van Tiegham cells were inverted after 2b min and aggregation was followed microscopically, on the inverted coverslips.

Cell breakage and enzyme assays Cells (10 ml of 10’ cells/ml) were harvested bv centrifugation at 700 g, washed in buffer (1 x l& M Tris-Cl, pH 7.4; 2 x 1O-2 M MgSO& and the cell pellets stored at - 70°C until next day. The cell pellets were then resuspended in 1 x lo-* M Tris-Cl, pH 7.5; 2 x lo-* M MgS04 and the cells disrupted by freezing and thawing until cell breakage was complete, as assessedmicroscopically. CAMP-dependent phosphodiesterase activity of the cell extracts (200 pg protein) was determined [12].

RESULTS Factors affecting the agglutination of D. discoideum by ConA

It has been shown recently that cells of D. discoideum are agglutinated by Con A [5]. In

order to fully characterize this agglutination, the effect of varying the incubation conditions Washed cells were resuspended in water at a cell density of 1.5 x lo8 cells/ml and 0.3 ml was disnensed was studied. onto ihe surface of a 42 mm Whatman No. 5b filter (a) Effect of incubation time: In all the paper, saturated with lower pad solution (KCl, 2 x 10-e M; MgC&, 3 x 1O-a M; phosphate, pH 6.5, previously reported experiments, a 10 min 5 x lo-* M and streptomycin sulphate, 500 mg/l), incubation time was used [5] since this time inside 60 mm plastic Petri plates. The Petri plates were incubated at 22°C and the developmental time gave highly reproducible results. As shown sequence progressed as previously described [l 11. in fig. 1, a 10 min incubation is sufficient Several methods were used to investigate the ef.to give maximum agglutination at all levels fects of ConA on differentiation. (a) Washed cells were resuspended in buffered of ConA used. In fact, longer incubation Bonner’s salts solution at a density of 1.5 x 108cells/ml times tend to slightly reduce the extent cl’ and incubated in the presence of 110 pmoles/ml NaCl

Differentiation

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agglutination. This experiment also clearly Table 1. Effect of cell density on ConAshows the background agglutination in the dependent agglutination of D. discoideum absence of Con A. This background aggluti- Ax-2 nation was subtracted from the total agglutiConA-dependent agglutination&’ nation observed in most assays to give the Cell densitya (cell no./ml) ( %) ConA dependent agglutination as described previously [5]. 8.0 x lo5 62 (b) Effect of cell density: Within the range 1.6x 1~ x lo6 ii of 1.5-3.5 X lo6 cells/ml agglutination was 2.6 3.5 x 106 88 68 unaffected by cell density. However, as shown 5.2 x 106 in table 1, at a cell density of 8 x lo5 cells/ml a Washed cells of strain Ax-2 resuspended in buffered and 5.2 X lo6 cells/ml agglutination was Bonner’s salts solution at the indicated cell density. slightly reduced. All subsequent assays were b Agglutination due to incubation in the presence of 1.6 pg/ml ConA for 10 min at 22°C and 200 rpm. conducted at cell densities between 1.5 and All assays were corrected for background agglutination in the absence of Con A [5]. 3.5 X lo6 cells/ml. (c) Effect of gyration speed: Many plant lectin agglutination assays have been con- D. discoideum at these speeds.All agglutinaducted on gyratory shakers at speedsbetween tion assays were therefore conducted at 200 60-80 rpm [3, 13, 141. However, there is rpm. Moreover the higher gyratory rate is very little ConA induced agglutination of useful in that it reduces background agglutination in the absenceof ConA. (d) Effect of incubation media: All preliminary agglutination studies [5] were conducted in buffered Bonner’s salts solution [lo], a standard salts solution for the resuspension of D. discoideum myxamoebae. Considerable background agglutination was always observed under these conditions (fig. 1, table 2). Incubation of cells in a buffered salts solution that contained EDTA and no divalent cations resulted in considerable reduction in the background agglutination, but it now required 4-5 times higher ConA concentrations to bring about the same agglutination (table 2). Fig. 1. Abscissa: time (min); ordinate: no. of unSince we were interested in subsequently agglutinated cells x 1O-e/ml. Time course of ConA-mediated agglutination of testing the effects of ConA on differentiacells of D. discoideum, AX-~. Cells were harvested tion, we also tested the Con A induced agglufrom the exponential phase of growth, washed and resuspended hi buffered Banner’s salts solution at a tination of cells resuspended in lower pad cell density of 2.4 x ll)d cells/ml. At zero time, 110 solution [ 111. Con A mediated agglutination ,umoles of NaCl and, in addition: 0, no ConA; 0, 2.0 pg ConA; l , 4.0 pg ConA; n , 8.0 pug ConA of cells resuspended in lower pad solution were added to 5 ml aliquots of cell suspension. Inoccurred only at high ConA concentrations, cubations were at 22°C and at 200 rpm on a gyratory shaker, At the indicated times, small aliquots and there was no background agglutination were withdrawn and the number’ of una%glutidated under these conditions. cells was determined as described in the text. Exptl Cell Res 92 (1975)

Cell surface changes during differentiation

Table 2. Effects of various incubation media on the agglutination of D. discoideum by C6n A

(e) Effect agglutination:

315

of sugars on Con A-mediated

Agglutination of a variety of cell types by ConA is totally inhibited by Agglutinated sugars with glucosidic and mannosidic linkIncubation mediaa cellsc ( X) Strain ages [l], and, as expected, the Con A induced agglutination of D. discoideum was completely buffered Bonner’s salts Ax-2 solution,b inhibited by a 10 min pre-incubation in the no ConA presence of 4 x lo4 M a-methyl glucoside, + 0.4 ,ug/ml Con A + 0.8 ,ug/ml Con A 1O-3 M maltose or 1O-3 M trehalose. In + 1A ,ug/ml Con A further agreement with anticipated results, + 80 pg/ml succ-Con A pre-incubation of cells with 2 x 1O-3 M gluNC-4 buffered Bonner’s salts solution,b cose, 2-deoxyglucose, 3-O-methyl glucose, 13 no ConA fucose, arabinose, galactose, N-acetyl galac+ 4 &ml ConA 52 + 8 ,ug/ml ConA 79 tosamine or mannose did not prevent sub+ 16 uelml ConA 95 sequent agglutination by Con A. However, + 80 yg/ml succ-Con A 12 although the sensitive precipitin assay [15] Ax-2 buffered salts - EDTA,’ no ConA 0 demonstrated that cc-glycosidic linkages are + 1.6 rg/ml Con A 21 considerably more effective inhibitors of + 3.2 ,ug/ml Con A 5.5 Con A-to-polysaccharide binding than fiAx-2 lower pad solutioqd no ConA 0 glycosidic linkages, ConA mediated agglu+ 8 rg/ml Con A tination of D. discoideum was inhibited to the + 16 pg/ml ConA ii + 40 pg/ml Con A 96 same extent by 1O-3 M a-methyl glucoside, p-methyl glucoside or cellobiose. a All incubation media contained 22 ~moles/ml NaCl Background agglutination, i.e. agglutinain addition to the listed components. b KCl, 1 x 10-e M; NaCI, 1 x lo-* M; CaCl,, 1.5 x tion in the absence of ConA, is completely lo-* M; Tris-Cl (pH 7.5), 1 x 1O-s M. inhibited by pre-incubation of cells with ’ KCl, 1 x 10-a M; NaCl, 1 x 1tF M; EDTA, 1 x 1O-4M; Tris-Cl (pH 7.5), 1 x lo-’ M. 1O-3 glucose, maltose, sucrose, trehalose or d KCl, 1.5 g/l; MgC&* 6H,O, 0.5 g/l; streptomycin cellobiose, was partially inhibited by presulphate, 0.5 g/l; phosphate buffer (pH 6.5), 0.05 M. e The number of unagglutinated cells was determined. incubation with 1O-3 M E-methyl glucoside The data is expressed as the percentage of agglutinated or p-methyl glucoside, and was unaffected cells, relative to the original cell number. by pre-incubation with 5 x 1O-3 M 3-OData also presented in table 2 shows that methyl glucose, galactose, mannose, fucose, the wild-type strain NC-4 is also agglutinated N-acetyl galactosamine or fructose. This data by Con A but considerably more Con A is indicates that an intact glucose residue is the required for agglutination of this strain than fundamental structure involved in background for AX-~. The background agglutination of agglutination, whereas the previous data NC-4 under these incubation conditions was shows that ConA agglutination involves far less than that of Ax-2 (table 2). Since the glycosidic linkages. growth of NC-4 to confluence produces a (f) Effect of temperature on ConA agglutimixed harvest of log, late log, and stationary nation: The agglutination of D. discoideum phase cells, these differences in agglutination by Con A is markedly affected by temperamay be more apparent than real and could, ture. As shown in table 3, there is very little in fact, reflect the growth phase of the cells cell agglutination at 4°C at concentrations of tested (cf fig. 4). Con A that bring about marked agglutination I

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Table 3. The effect of temperature on the agglutination of cells of D. discoideum Ax-2 by Con A Incubation Con A cont. Con A dependent agglutination’ ( %) temp. CC) Cudml) 22 37 4

0.8 1.6 0.8 1.6 0.8 1.6 16.0

70

93 68

95 7

it:

a Corrected for background agglutination in the absenceof ConA. at 22” or 37°C. Background agglutination was eliminated by incubation at 4°C. There is no unique transition temperature below which agglutination does not occur. As shown in fig. 2, the transition temperature is dramatically influenced by the concentration of ConA. A similar effect is also shown in table 3, where incubation at 4°C in the presence of elevated levels of ConA does cause marked agglutination. (g) Effect of inhibitors on Con A agglutination: Preincubation of cells in the presence of metabolic inhibitors sodium azide and dinitrophenol prior to the addition of Con A markedly inhibited agglutination, as shown in table 4. Also shown in table 4 are results of an experiment in which cells were preincubated in the presence of 80 ,ug/ml succConA for 10 min prior to addition of ConA. Succ-Con A at this concentration produces no agglutination (table 2), and addition of ConA to the treated cell suspension failed to produce any agglutination, suggesting that ‘univalent’ Con A binds to cells and prevents the subsequent binding of native ConA. Detection of cell surface changes during differentiation by Con A agglutination It was shown previously [5] that dissociated ‘aggregation phase’ cells of strain Ax-2 were Exptl CeN Res 92 (197.5)

Fig. 2. Abscissa: incubation temp. (“C); ordinate:

ConA dependentagglutination ( %). Temperature dependenceof ConA-mediated agglutination of cells of D. discoideum, NC4. Cells were harvestedfrom confluent growth plates, washed and resuspendedin buffered Bonner’s salts solution at a cell density of 2.8 x 10’ cells/ml. Aliquots (5 ml) were incubated at the indicated temperaturesin the presenceof 110 rmoles NaCl and either 0, 40; or o 60 pg ConA. After 10 min agglutination was assessedas describedin the text. far less susceptible to Con A agglutination than were growing cells. A similar result was obtained with cells of strain NC-4 (unpublished observations). In order to precisely monitor this phenomenon for strain AX-~, differentiation filters were set up and cells washed off after known intervals and tested for their agglutination response to Con A. Table 4. The effect of preincubation of cells of D. discoideum NC-4 in the presence of possible inhibitors Pre-incubation conditionsa

Con A dependent agglutin&on ( %)b

No addition + 2.5 x lo-* M sodium azide + 2.0 x 1O-aM dinitrophenol + 400yg succ-ConA

92.5 18 15 2

a Five ml aliquots of cell suspensionwerepreincubated in the presenceof the various components for 10 min at 22°C and 110min. b ConA (80 pg) and NaCl (110pmoles) were added and agglutination was determined after a further 10 min incubation. All values are correctedfor background agglutination in the absenceof Con A.

Cell surface changes during differentiation

317

this laboratory that cells grown well into the stationary phase of growth fail to differentiate, it is ‘possible that the cell surface site responsible for this background agglutination is necessaryfor aggregation. However, the loss of background agglutination occurs considerably sooner than the loss of capacity to differentiate (unpublished observations). Effects of ConA on differentiation

5

15

10

Fig. 3. Abscissa: time (hours); ordinate: ConA cont. required to produce 50 % agglutination @g/ml). Con A concentration required for 50 % agglutination of cells of D. discoideum Ax-2 at various stages of differentiation. Cells were allowed to differentiate on Whatman No. 50 filter papers. Under these conditions arrareaatina streams were visible at 8 h. ‘slug’ forma&-occuked at 16 h. Cells were ha& vested into buffered Bonner’s salts after the indicated time periods of differentiation, dissociated by tituration and vortex treatment, and tested for ConA dependent agglutination, as described in the text.

The data in fig. 3 demonstrates a gradual change in the responseof these cells to Con A. Detection of cell surface changes during transition from exponential phase to stationary phase

growth

It was shown previously that cells in the stationary phase of growth were also less susceptible to agglutination by Con A than exponentially growing cells [5]. To study the onset and degree of the cell surface change during this transition, cells were grown for increasing time periods, harvested, washed, and tested for ConA dependent agglutination (fig. 4). There was a gradual decreasein the susceptibility of cells to ConA agglutination, as cells passed into stationary phase (fig. 4). During the course of these studies, it was observed that as cells passedfrom exponential phase to stationary phase of growth, background agglutination decreased markedly. Since it has been consistently observed in

of

D. discoideum Ax-2 It is conceivable that Con A binding sites are necessary for aggregation and subsequent normal differentiation, and that blocking them with ConA might have aberrant effects on the process.In order to test this possibility, cells were set up to differentiate in the presence of the lectin. The experiment was performed a number of ways. Cells were incubated in the presence of various concentrations of Con A (50-800 ,ug/ ml) for 10 min and then allowed to differentiate on Whatman No. 50 filter papers. Al-

1 Fig. 4. Abscissa: time (hours); ordinate: (left) cell no. x 10-6/ml; (right) ConA cont. required to produce 50 % agglutination (uglml). Con A concentration ~e$&d for 50 % agglutination of cells of D. discoideum Ax-2 at various times during the transition from ‘exponential phase’ of growth to ‘stationary phase’ of growth. Cells were harvested at the indicated growth times, having attained the indicated cell density (0). Cells were washed, resuspended in buffered Bonner’s salts solution and tested for Con A dependent agglutination as described in the text (0). Exptl Cell Res 92 (197.5)

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though agglutination was obtained at all concentrations there was no effect on the time course of differentiation. Since it was conceivable that the failure of ConA to affect differentiation was due to its interaction with the @-glucoside residue of the cellulose filter paper, effectively removing Con A from the cell surface, alternative substrata for the differentiation process were employed. Cells were incubated with ConA in Van Tiegham cells [lo] containing Bonner’s salts solution, NaCl, and various concentrations of ConA (1.6-160 ,ug/ml). Incubation in the presence of 80 and 160 ,ug/ml Con A indirectly prevented aggregation after inversion of the Van Tiegham cell by producing such massive agglutinates that they fell off the coverslip. ConA at concentrations of 1.616 pg/ml had no effect on differentiation. Cells were also preincubated in the presence of the various concentrations of ConA at 22°C and 200 rpm on a gyratory shaker, and then added to Van Tiegham cells. This procedural variation gave identical results. In another experiment, cells were incubated with ConA and then layered onto the surface of 2 % agar Petri plates. Again no effect was observed on differentiation at ConA concentrations as high as 800 pg/ml. While these studies were in progress, a report appeared which described the inhibitory effect of ConA on the time course of differentiation of D. discoideum NC-4 [16]. These workers added drops of cell suspension to the surface of agar containing various concentrations of ConA, a modification of a previously published procedure [17]. We repeated this procedure with strain -4x-2, but obtained inconsistent results due to unequal drying of the drops. When 0.2 ml of cell suspension was spread on the surface of the 2 % agar Petri plate, the following consistent results were obtained. The aggreExptl Cell Res 92 (197.5)

gation of cells spread on agar containing 500 ,ug/ml ConA was delayed between 6-8 h. Lower concentrations of Con A (100 pug/ml, 200 pg/ml) had no effect on the time course of aggregation. Although there was an inhibitory effect of ConA it was less pronounced than that reported previously [16]. Effect of ConA on differentiation

of

D. discoideum NC-4 An important difference between the studies described above and those reported by Gillette & Filosa [16] was their use of the wild type strain NC-4, whereas we used the axenic mutant, AX-~. Since the Con A agglutination studies reported here had already indicated possible cell surface differences between the two strains, we decided to study the ConA effect on the differentiation of the wild type strain. Cells were harvested from confluent growth plates, washed, resuspendedin buffered Bonner’s salts solution and incubated in the presence of NaCl and various Con A concentrations as described under Methods. In some experiments cells were layered onto Whatman No. 50 filter papers and incubated at 22°C. Preincubation in the presence of Con A concentrations as high as 800 pg/ml had no adverse effect on differentiation, although massive agglutination occurred during the preincubation. In other experiments cells preincubated with ConA were layered onto agar plates and allowed to differentiate. Again there was no effect of ConA on the time course of differentiation, even at ConA concentrations of 640 ,ug/ml. In a third series of experiments cells preincubated with ConA were placed in Van Tiegham cells. ConA at a concentration of 1.6 ,ug/ml, the lowest concentration tested, delayed aggregation 8 h. Con A at a concentration of 16 ,ug/ml delayed aggregation 24 h. Results using higher concentrations of ConA were irrel-

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319

cells and was found to have no effect on the time course of aggregation at a concentration of 160 pg/ml. Effect of Con A agglutination on subsequent differentiation of dissociated aggregates of D. discoideum Ax-2

Cells were allowed to differentiate on Whatman filter discs for 15 h after which time they had formed distinct aggregates.These aggregates were harvested into buffered Bonner’s salts solution at a cell density of 5 x 10’ Fig. 5. Abscissa: time (hours); ordinate: CAMP spe- cells/ml. cific phosphodiesterase activity (nmoles, min-I, mg-9. The cell suspensionswere incubated for 10 Washed cells of D. discoideum, Ax-Z, harvested from the exponential phase of growth, were resuspend- min in the presenceof up to 100,ug/ml ConA ed in buffered Bonner’s salts solution at a cell density of 10’ cells/ml. Sixty ml of cell suspension was in- and then 0.2 ml was dispensed onto 2 % agar cubated in a 250 ml Erlenmeyer flask at 22°C and plates. Under these conditions, control cells 200 rpm on a gyratory shaker, in the presence of 22 incubated in the absence of Con A reaggre~moles/ml NaCl and either (0) no Con A, or (0) 160 pg/ml ConA. After the indicated time periods, gated in 4 h, while cells that were incubated cells were harvested, washed, and cell-free extracts prepared as described under Methods. Phospho- in the presence of 100 pg/ml Con A were die&erase activity was determined as described pre- slightly delayed, reaggregating in about 6 h. viously [12].

evant, since the large agglutinates resulting from incubation in the presence of Con A fell off the coverslips after inversion of the Van Tiegham cells. Finally cells of NC-4 were layered onto the surface of 2 % agar plates containing various concentrations of Con A dissolved in the agar. Aggregation was delayed at all the tested levels of Con A. The delay was 8 h at 20 pg/ml ConA, the lowest level of Con A tested, and 30 h at 800 pg/ml Con A, results similar to those of Gillette & Filosa

WI. Effect of succinylated-Con A on differentiation of D. discoideum NC-4

Succinylated-ConA (succ-ConA) binds to the cell surface at the ConA binding site, but does not bring about agglutination (table 2). The effect of succ-ConA on the differentiation of strain NC-4 was tested in VanlTiegham

Effect of Con A on phosphodiesterase activity of D. discoideum Ax-2

Gillette & Filosa [16] have suggestedthat the delay of aggregation by Con A is due to an interference in the CAMP chemotactic system. They showed that incubation of cells in the presence of ConA markedly increased the activity of the CAMP specific phosphodiesterase. Since we found that ConA had a far less pronounced effect on the aggregation of strain AX-~, it might be anticipated that it would also have a less pronounced effect on the phosphodiesterase. Cells of Ax-2 were therefore incubated in the presence of Con A (160 pg/ml) and phosphodiesterase activity was determined in cell extracts. The data from this experiment is shown in fig. 5. In control incubation (no ConA) there was a gradual increase in phosphodiesterase activity over a period of several hours, a result similar to that obtained by earlier Exptl Cell Res 92 (1975)

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workers [12]. However, in the presence of ConA a large increase in activity was observed after only 1 h incubation. The activity then decreased during the remainder of the incubation period. Gillette & Filosa also observed a large increase on the phosphodiesterase activity of strain NC-4 during the first hour of incubation in the presence of ConA. Thus although Con A has no effect on aggregation of Ax-2 at this concentration (160 ,ug/ml), it produces a large increase in phosphodiesterase activity. DISCUSSION A simple microscopic assay was used in a previous communication [5] to determine the agglutination of cells of Dictyostelium discoideum Ax-2 by ConA. A 10 min incubation period in the presence of Con A was found to be highly reproducible and was used throughout the study [5]. However, recently it has been reported that the Con A mediated agglutination of teratoma cells and dissociated seaurchin embryo cells [14] and human erythrocytes [18] required as long as 60 min incubation before optimum results were achieved, and it was suggestedthat many of the earlier studies which relied on short incubation periods should be re-evaluated. The data presented in this paper, however, shows that for the ConA agglutination of D. discoideum cells, a 10 min incubation period is sufficient. In fact prolonged incubation (fig. 1) leads to a slight reduction in agglutination, despite the fact that there is no decrease in the binding of 1251-ConA to the cell surface during this time period (unpublished observations). Previous studies have shown that the Con A agglutination of a variety of mammalian cells is temperature dependent [20-241. Agglutination occurs at 22 and 37°C but not at 0-4°C. Furthermore, there is a clustering of ConA Exptl CeN Res 92 (197.5)

binding sites at 22 and 37°C but not at 4°C consistent with the idea that clustering of binding sites is essential for agglutination. Noonan & Burger [24] have studied in detail the temperature dependence of the ConA agglutination of polyoma transformed 3T3 cells and have found that cells are agglutinated above 18”C, partially agglutinated between 10 and 18°C and not agglutinated below 10°C. They suggest that the temperature profile of the transition from unagglutinated to fully agglutinated states may reflect a phase transition in the membrane lipid. Detailed temperature dependence studies are reported here for the ConA agglutination of D. discoideum and similarly sharp temperature transitions are observed. However, the temperature transition is markedly dependent upon ConA concentration, with a lower transition temperature being observed at higher concentrations of ConA (fig. 2). In fact agglutination was observed even at 0°C if a sufficiently high Con A concentration was employed (table 3). Thus high concentrations of lectin appear to be able to influence a phenomenon which should only reflect the thermodynamic state of the membrane. This effect may indicate that the Con A concentration that is necessary for agglutination is far below the concentration required to fully saturate the available cell surface binding sites. At normal physiological temperatures, only a few of the potential sites would be occupied, but clustering and thus agglutination could occur due to the high fluidity of the membrane. At low temperatures the sameconcentration of Con A would again generate low density of occupied binding sites but the decreasedfluidity of the membrane would prevent both clustering and agglutination. Elevated Con A concentrations, however, could generate local regions with a high density of occupied sites thus mimicking the clustering of such sites despite

Cell surface changes during differentiation

the low fluidity of the membrane lipid, The binding of ConA to the cell surface is currently being studied to test this possibility. It should be emphasized that the lack of agglutination at 0°C is not a decreased rate phenomenon. There is no agglutination even after prolonged incubation at low ConA concentration. Agglutination of cells of D. discoideum by ConA is prevented if cells are preincubated with the metabolic inhibitors sodium azide and dinitrophenol. There have been reports that the lectin induced agglutination of lymphocytes [25] and rat ascites tumour cells [26] is also inhibited by metabolic inhibitors, and, although precise nature of the inhibition is not known, an involvement of microtuble protein in the clustering of ConA binding sites has been invoked [271.In other studies metabolic inhibitors had no effect on the agglutination of Ehrlich ascites tumour cells [23] and polyoma transformed 3T3 cells [24], so that the requirement of metabolic energy for agglutination may not apply to all cells. There is a gradual decreasein the susceptibility of cells of D. discoideum to agglutination by ConA, as they pass from the ‘growth phase’ to the ‘aggregation phase’ during normal differentiation. Our quantitative observations (fig. 3) are substantiated by the recent scanning electron micrographs of the surface of D. discoideum agglutinated by Con A [28]. There is a distinct difference in the appearance of ConA treated vegetative cells and Con A treated aggregation phase cells [28]. Although the time course of the change in susceptibility to ConA is similar to the time course of the formation of aggregation competence (site A contacts) described by Gerisch and co-workers [19], there is no evidence to suggest that ConA binding sites are involved in the normal aggregation process.In the present study, in fact, ConA and succ-Con A were found to have very little

381

effect on the differentiation of strain Ax-2 of Dict&wtelium discoideum, suggesting that ConA binding sites are not involved directly in the normal aggregation process. Steinberg & Gepner [13] came to similar conclusions from studies on the tissue specific reaggregation and cell sorting of mixed chick heart and retinal cells. However, since it has been shown that ConA is internalized in some cell types [29], it is possible that occupied ConA binding sites are removed from the surface and replaced by newly synthesized sites that are involved in cell-cell interaction. This possibility is currently under study in this laboratory. It must be pointed out that Gillette & Filosa [16] did observe a considerable delay in aggregation of the wild type stain of Dictyostelium discoideum NC-I, if these cells were allowed to differentiate on agar containing high concentrations of ConA and we have confirmed their observations. However, in identical experiments with AX-~, the delay in aggregation was far less pronounced. Thus clearly there is a difference between the axenic strain Ax-2 and its wild-type parent strain NC-4 with regard to the effect of Con A on differentiation, but this very strain difference makes it difficult to believe that ConA binding sites are vitally involved in differentiation. Furthermore succ-ConA, which prevents ConA agglutination and therefore presumably occupies the same binding site, has no effect on the differentiation of strain NC-4. Gillette & Filosa have suggestedthat ConA interferes with the rate of aggregation of strain NC-4 by interfering with the mechanism of CAMP chemotaxis [16]. Specifically they showed that ConA caused a rapid increase in the activity of CAMP specific phosphodiesterase. However, more work will be needed to substantiate this suggestion. We find that ConA markedly stimulates the CAMP phosphodiesterase activity of strain Exptl Cell Res 92 (1975)

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Weeks and Weeks

AX-~, while having only a slight effect on the time course of differentiation of this strain. There is clearly a marked change in susceptibility to ConA agglutination as cells go through the differentiation process. This change also occurs as cells pass from exponential phase of growth into stationary phase, and it is conceivable that the change in ConA agglutination reflects a difference in surface architecture between the growing and the non-growing states. This suggestion may explain the similarities in patterns of ConA agglutination that have been observed in other differentiating systems [3, 4, 61 where cells may be undergoing a change from rapidly growing to slowly growing states. In this connection it is interesting that cells are more readily agglutinated during mitosis [30] than at other times in the cell cycle and malignant cells are far more susceptible to agglutination by ConA than are their normal counterparts.

7. Sussman, M, Methods in cell physiology (ed D M Prescott) vol. 2, p. 397. Academic Press, New York (1966). 8. Watts, D J & Ashworth, J M, Biochem j 119 (1970) 171. 9. Gunther, G h, . . cu.& .I LL, I OLl~la, I, Lu,ilr,Ll~ham, B A & Edelman, G M, Proc natl acad sci us 70 (1973) 1012. 10. Bonner, J T, J exptl zoo1 106 (1947) 1. 11. Ellingson, J S, Telser, A & Sussman, M, Biochim biophys acta 244 (1971) 388. 12. Malchow, D, Nagele, B, Schwarz, H & Gerisch, G, Eur j biochem 28 (1972) 136. 13. Steinberg, M S & Gepner, I A, Nature 241 (1973) 249. 14. Oppenheimer, S B & Odencrantz, J, Exptl cell res 73 (1972) 475. 15. So, L L & Goldstein, I J, Biochim biophys acta 165 (1968) 398. 16. Gillette, M U & Filosa, M F, Biochem biophys res commun 53 (1973) 1159. 17. Konijn, T M & Raper, K B, Dev biol 3 (1961) 725. 18. Gordon, J A & Marquardt, M D, Biochim biophys acta 332 (1974) 136. 19. Beug, H, Katz, F E & Gerisch, G, J cell biol 56 (1973) 647. 20. Inbar, M, Ben-Bassat, H & Sachs, L, Proc natl acad sci US 68 (1971) 2748. 21. Nicolson, G L, Nature new biol 243 (1973) 218. 22. Inbar, M, Huet, C, Oseroff, A R, Ben-Bassat, H & Sachs, L, Biochim biophys acta 311 (1973) 594. 23. Inoue, M, J cell sci 14 (1974) 197. 24. Noonan, K D &Burger, M M, J cell bio159 (1973)

This work was supported by grants from the National Research Council of Canada and the National Cancer Institute of Canada.

25. Loor, F, Exptl cell res 82 (1973) 415. 26. Kaneko, I, Satoh, H & Ukita, T, Biochem biophys res commun 48 (1973) 1504. 27. Yin, H H, Ukena, T E & Berlin, R D, Science 178 (1972) 867. 28. Rossomando, E F, Steffek, A J, Mujwid, D K & & Aleksander, S, Exptl cell res 85 (1974) 73. 29. Barat, N & Avrameas, S, Exptl cell res 76 (1973) 451. 30. Burger, M M, Fed proc 32 (1973) 91.

REFERENCES 1. Sharon, N & Lis, H, Science 177 (1972) 949. 2. Moscona, A A, Science 171 (1971) 905. 3. Kleinschuster, S J & Moscona, A A, Exptl cell res 70 (1972) 397. 4. Weiser, M B, Science 177 (1972) 525. 5. Weeks, G, Exptl cell res 76 (1973) 467. 6. Krach, S W, Green, A, Nicolson, G L & Oppenheimer, S B, Exptl cell res 84 (1974) 191.

Exptl Cell Res 92 (1975)

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Received August 27, 1974 Revised version received November 5, 1974