Printed in Sweden Copyright @ 1978 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/78/1171-0121$02.00/0

Experimental Cell Research 117 (1978) 121-126 THE EFFECT

OF CYTOCHALASIN

ON LECTIN-MEDIATED EARLY

EMBRYONIC

J. ROBERT PHILLIPS

B AND COLCHICINE

AGGLUTINATION CHICK

OF

CELLS

and SARA E. ZALIK

Department of Zoology, University of Alberta, Edmonton, Alberta, Canada T6G 2E9

SUMMARY The effects of cytochalasin B (CB) and colchicine on the lectin-mediated agglutination of dissociated cells from chick embryos at the early primitive streak stage were studied. Cells incubated in the absence of the above-mentioned drugs were agglutinable with concanavalin A (ConA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA). A pre-incubation with neuraminidase was required to render the cells agglutinable with soybean agglutinin (SBA). This treatment had no appreciable effect on the agglutinability of the cells with the other three lectins. Treatment with the drug colchicine had no appreciable effect on the extent of agglutination with any of the above-mentioned lectins. Cells treated with CB dissolved in dimethylsulfoxide (DMSO) in saline, exhibited a reduced lectin-mediated agglutinability. However, a similar decline in agglutinability was observed in controls incubated in saline containing DMSO alone. It is suggested that structures sensitive to colchicine and CB do not play a major role in the control of surface lectin receptors in early embryonic cells.

There is evidence to suggest that cell surface carbohydrates may be involved in cell recognition and adhesion [l-3]. Lectins, proteins which bind specific carbohydrate groups [4],’ have proven useful as probes in the investigation of these cell surface components. One of the consequences of lectin binding to dissociated early embryonic cells is the agglutination of cells. It has been suggested that the agglutination reaction is due to a topographical change in the distribution of lectin receptors at the cell surface [5, 61. In addition, it has been proposed that the mobility and distribution of these receptors is under the control of a plasma membraneassociated cytoskeleton composed of microtubules and microfilaments [7]. The evidence for cytoplasmic control of membrane receptor mobility comes from experiments in which colchicine, an alkaloid which prevents polymerization of microtubule subunits [8], was found to alter the extent of

lectin-induced agglutination, as well as the distribution of receptors at the cell surface [6, 9-121. Likewise, CB, which disrupts actin-containing microfilaments [ 13, 141, has been reported to alter the redistribution of surface receptors [9-121. Previously, we have reported that early embryonic cells are agglutinated with concanavalin A (ConA), wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA), and soybean agglutinin (SBA) [IS171. In this study we tried to determine whether cytoskeletal elements are involved in lectin-mediated agglutination; to this purpose the effects of CB and colchicine on this process were studied. In addition, some observations on cell morphology are reported. MATERIALS

AND METHODS

Chick embryos at the early primitive streak stage (stage 3-4, Hamburger & Hamilton) [ 181were excised from the yolk and dissected free of the vitelline memExp Cell Res 117 (1978)

122

Phillips

and Zalik

brane in Pannett & Compton’s saline (PCS) [ 191,with 15 mM Hepes (Sigma), (pH 7.5). Adhering yolk was removed from the blastoderm by gently rinsing the embryos in numerous changes of PCS. The blastoderms were dissociated in cold (4°C) Ca2+-, Mg2+-free PCS, pH 7.8, by pipetting through three Pasteur pinettes of decreasing bore size (rangina from 3 to 0.5 mm internal diameter). This technique resulted in a cell suspension containing greater than 75% single cells with most remaining clumps containing fewer than 10 cells. Dye exclusion studies with Trypan blue revealed that 95 % of the cells remained unstained. The cell suspension was centrifuged for 12 min at 19 g in a Clinical Centrifuge and the resulting cell pellet resuspended in complete PCS to a final concentration of 1X lo6 cells/ml. Cell suspensions in PCS were incubated at room temperature for 30 min in the presence of either 60 PM colchicine (Sigma) or 40 PM CB (Aldrich), or in the simultaneous presence of cytochalasin and colchitine. Initially, a stock solution of cytochalasin was made up in dimethylsulfoxide (DMSO) such that when added to the cells the DMSO concentration was 2% (v/v); in subsequent experiments the concentration of DMSO was reduced to 0.5%. Control incubations with DMSO at the same concentrations as in the cytochalasin treatment were also performed. The effects of cytochalasin were the same at both DMSO concentrations. Neuraminidase treatment of cells was performed by incubatine 1x 106cells/ml with 10 units of neuraminidase (from Vihrio co&a Behringwerke, AC) in PCS for 30 min at 37°C. This neuraminidase orenaration is stated by the manufacturer to be free of protease, aldolase and lecithinase activity and no proteolytic activity can be detected in 50 units of this enzyme using Azocoll (Calbiochem) as substrate. At the end of the incubation, cells were washed with cold PCS and resusnended ih fresh PCS. The lectins utilized in this study were ConA (Pharmacia), WGA (Miles), RCA, and SBA. The latter two lectins.were a gift from Dr G. M. W. Cook of the Department of Pharmacology, Cambridge, UK. Agglutination assays were performed within porcelain rings mounted on glass slides. The desired concentration of lectin was obtained by placing appropriate amounts of a stock lectin solution and saline on the slide. Approx. 3 x lo4 cells were added to make a final volume of 50 ~1. The plates were incubated at room temperature with periodic swirling for 30 min before scoring agglutination. Agglutination could usually be distinguished within 10 min but for a more consistent evaluation it was scored at 30 min. Longer incubation periods resulted in slight aggregation occurring which interfered with the evaluation of agglutination; although aggregation could usually be distinguished from agglutination because cell aggregates tend to be more compact and regular in outline than agglutinated clumps of cells. Agglutination was assessed qualitatively on a scale of 0, +, ++, +++, and ++++, where 0 indicates no aaelutination. + indicates a 30% increase in agglutinated cells, +i is used when 3(Mo% agglutinated cells are present, + + + indicates 60-90 % @glutinated cells and + + + + indicates above 90% of a& glutination. Agglutination was observed with a dissect-

ing microscope taking into account the number and sizes of agglutinated clumps as well as the number of non-agglutinated cells. In addition, cell morphology was observed under the phase contrast microscope.

RESULTS Maximal and consistent agglutination of dissociated blastoderm cells was observed at 10 pg/ml ConA (table 1); a lower and less consistent agglutination response was observed with lower concentrations of this lectin. Maximal agglutination with WGA occurred at a concentration of 10 pglml, although slight agglutination occurred at lower concentrations (table 2). The agglutination response to the RCA preparation was generally lower than that of WGA or ConA but it was consistent over the concentrations of lectin tested (table 2). SBA, at concentrations as high as 150 pg/ml, did not induce agglutination, however, treatment with neuraminidase for 30 min at 37°C rendered the cells agglutinable with 50 pg/ ml SBA (table 1). The neuraminidase incubation did not appreciably alter the agglutinability of the cells with the other three lectins. Free yolk particles did not agglutinate with any of the lectins at concentrations which caused cell agglutination. DMSO, DMSO plus CB, and colchicine had no effect on ConA or SBA mediated agglutination (table 1). A lack of effect on ConA-induced agglutination was also observed when combinations of colchicine and cytochalasin were used. DMSO, cytochalasin, and colchicine were not able to induce SBA agglutination in cells which had not been incubated with neuraminidase. The SBA-mediated agglutination, induced by neuraminidase treatment, was not affected by incubation with the abovementioned drugs. DMSO consistently caused a slight decrease in the agglutination induced by WGA

Cytochalasin and colchicine on embryonic cell agglutination

123

Table 1. Effect of DMSO, CB, and colchicine on ConA and SBA-mediated agglutination Lectin ConA

0 5 7.5 10 20 0 50 100 150 0 50 100

SBA

SBA” (neuraminidase treated cells)

(4)” (3) (2) (4) (3) (4) (4)

(2) (1) (4) (4)

(2)

Saline control

DMSO control 2-4 % (v/v)

CB 40 PM

Colchicine @PM

0 0 + +++ +++ 0 0 0 0 0 + ++

0 0 + +++ +++ 0 0 0 0 0 + ++

0 0 + +++ +++ 0 0 0 0 0 + ++

0 + +++ +++ 0 0 0 0 0 + ++

CB+ colchicine 8 +++

Cells were incubated in PCS in the absence or presence of DMSO, CB and/or colchicine for 30 min at room temperature. At the end of this period aliquots of the cell suspension were removed and incubated with lectin for 30 min at room temperature. Agglutination was assessed by the procedure described in Materials and Methods. LI Cells were incubated with 10 U/ml neuraminidase at 37°C for 30 min concomitantly with DMSO, CB or colchicine. b The numbers in parentheses refer to the number of experiments performed.

and RCA (table 2). When the results of the controls with these lectins, except with 20 CB effect on agglutination were compared pglml WGA where colchicine caused a with DMSO controls, no effect of this drug slight increase in agglutinability. Treatment was evident. However, at higher RCA con- with cytochalasin and colchicine combined centrations, treatment with CB brought ag- did not affect the agglutination induced by glutination up to the level of the saline con- these lectins. trol (table 2). Colchicine-treated cells were The effects of the various treatments on agglutinated to the same extent as the saline cell morphology were observed by phase Table 2. Effect of DMSO, CB, and colchicine on WGA and RCA-mediated agglutination Lectin WGA

Cont. (ccglml)

Saline control

DMSO control 24% (v/v)

CB 40 PM

Colchicine MPM

CB+ colchicine

0 2.5 5 ::

(4)= (2) (1) IZi

‘p; (1) (4)

0 + f ++ ++ 0 + + + +

0 + + ++ ++ 0 + + ++ ++

0 ++ ++ +++ ++++ 0 ++ + ++ ++

0

i.5 5

0 + ++ +++ +++ 0 ++ ++ ++ ++

RCA

:oo

(2)

+++ 0 ++

Cells were incubated in PCS in the absence or presence of DMSO, CB and/or colchicine for 30 min at room temperature. At the end of this period aliquots of the cell suspension were removed and incubated with lectin for 30 min at room temperature. Agglutination was assessed by the procedure described in Materials and Methods. 4 The numbers in parentheses refer to the number of experiments performed.

124

Phillips

and Zalik

Fig. 1. Chick blastoderm cells under phase contrast

microscopy. (a) Control cell; note hyaline lobopodial extensions (arrow), and abundance of yolk platelets in the cell’s cytoplasm; (b) cell in the presence of CB

contrast microscopy. Freshly dissociated cells suspended in PCS formed hyaline lobopodia (fig. 1a) which were observed to change their shape and position on the cell surface. Cytochalasin caused a decrease in the number of cells which formed lobopodia. This effect was apparent within 10 min after the addition of the drug to the suspension. Compared with the control cells, which had a smooth cell surface, many of the cytochalasin treated cells had a crenated cell surface (fig. 16). This was especially evident in the large yolky cells where it appeared as though the cell membrane had contracted around the cell, forcing the yolk platelets to bulge outwards. In addition, some cell lysis was apparent as evidenced by the increase in the abundance of free yolk platelets. Cells maintained in DMSO had a normal appearance; however, signs of crenation began to appear when cells were maintained in this medium for more than 45 min. Colchicine-treated cells had a similar appearance to control cells suspended in saline. Incubating the cell suspensions at 37°C tq,

Cd Rr\ //7 fIY7NJ

(40 PM). Note absence of lobopodia and the crenated cell surface. Two individual yolk platelets are in close proximity to the cell. Bar, 10 PM.

for neuraminidase treatment resulted in the formation of numerous small aggregates. This occurred in both the presence of neuraminidase and in saline controls, although as assessed visually, slightly more aggregation occurred in neuraminidase-treated cell suspensions. Concomitant incubation with 40 PM cytochalasin considerably decreased this aggregation; however, cells were still agglutinable with all four lectins. DISCUSSION The results presented here support previous findings [15, 161with regard to the agglutinability of chick blastoderm cells with ConA, WGA, RCA and the need for neuraminidase treatment to render the cells agglutinable with SBA. Although a slight variability in the agglutinability of different cell preparations existed, the same preparation was used for each series of experiments in which the effects of colchicine and cytochalasin on agglutination were assessed. In every case, control cell suspensions in the presence and

Cytochalasin and colchicine on embryonic cell agglutination absence of lectins were examined. Although the technique used in this study for assessing agglutination is subjective, the results reported here are valid as indicating some general characteristics of agglutinability. The experimental procedure used to dissociated chick blastoderms results in an abundance of yolk-laden hypoblast cells in the cell suspension, although considerable numbers of cells belonging to other cell populations within the embryo are also present [20]. Under the conditions used for assessing agglutination it was not possible to distinguish differential agglutinabilities among the cells in the suspension. Further experiments with cells isolated from different areas of the embryo are being performed to clarify this point. It has been proposed that cell surface receptor mobility may be controlled by a submembranous cytoskeleton sensitive to CB and colchicine [6, 71, and the mobility of lectin receptors has been implicated in lectin-mediated agglutination [5, 61. In the present investigation little effect of CB or colchicine was observed on lectin-mediated agglutination suggesting that, in these cells, the surface glycoprotein molecules, which act as lectin receptors and participate in an agglutination reaction, are not controlled to any appreciable extent by the microtubule, microfilament system. However, the possibility that the cytoskeletal elements of these early embryonic cells are more resistant to cytochalasin and colchicine treatment cannot be discarded. A similar lack of effect of the above-mentioned drugs on lectin-mediated agglutination has been reported for the early amphibian embryo [ 171, suggesting that the results mentioned herein are not restricted to cells of the chick embryo and may indicate a characteristic common to early embryonic cells.

125

An attractive possibility suggested by these results is that in early embryonic cells, which are frequently making and breaking cell contacts during morphogenesis, the cytoskeletal system plays a subordinate role to other means of cell surface receptor control. The local stabilization of phospholipids adjacent to these receptors could be involved; the decrease in agglutinability induced by DMSO, a stabilizer of phospholipid membranes [21, 221, suggests this possibility. In addition, the high affinity of the surfaces of these cells for divalent cations [23,24] may indicate a possible role of the latter in the control of cell surface receptors. Alternatively, the glycoprotein lectin receptors may be influenced by lectin-like molecules present either in adjacent cells or in the intercellular environment. The finding of lectin activity in extracts of chick blastoderms makes this an attractive possibility [25]. As differentiation proceeds and cells become committed or acquire permanent locations, the cytoskeletal elements of the cells may gradually take over control of receptor mobility, in conjunction perhaps with external elements at the cell surface. What relationship lectin receptors bear to the components at the cell surface which are involved in morphogenetic interactions remains to be established. This work was supported by the NC1 and in part by the NRC of Canada. We would like to thank Dr Nadine Miles for her helpful suggestions and discussion. We are also grateful to Mr W. Kulyk for his critical reading of the manuscript.

REFERENCES 1. Roseman, S, Chem phys lipids 5 (1970) 270. 2. Shur, B &Roth, S, Am zoo1 13 (1973) 1129. 3. Cook, G M W & Stoddart, R W, Surface carbohydrates of the eukaryotic cell. Academic Press, New York (1973). 4. Lis, H & Sharon, N, Ann rev biochem 42 (1973) 541. 5. Nicolson, G L, Int rev cytol39 (1976) 89.

126

Phillips

and Zalik

6. Poste, G, Papahadjopoulos, D & Nicolson, G L. Proc natl acad sci US 72 (1975) 4430. 7. Edelman. GM. Science 192(1976) 218. 8. Borisy, d G & Taylor, E W, J cell biol 34 (1967) 525. 9. Edelman, G M, Yahara, I & Wang, J, Proc natl acad sci US 70 (1973) 629. 10. McDonough, J & Lilien, J, J cell sci 19(1975) 357. 11. de Petris, S, Nature 250 (1976) 56. 12. Nicolson, G L & Poste, G, J supramol struct 5 (1976) 65 (39). 13. Lin, S & Spudich, J A, J supramol struct 2 (1974) 728. 14. Weihing, R R, J cell biol71 (1976) 303. 15. Zalik, S E & Cook, G M W, J cell biol 63 (1974) 385A. 16. - Biochim biophys acta 419 (1976) 119. 17. Fraser, B R & Zalik, S E, J cell sci 27 (1977) 227. 18. Hamburger, V & Hamilton, H L, J morph01 88 (1951) 49.

19. Pannett, C A & Compton, A, Lancet 206 (1924) 381. 20. Milos, N. Zalik, S E & Phillips, J R. In preparation. 21. Lyman, G H, Preisler, H D & Papahadjopoulos, D, Nature 262 (1976) 360. 22. Lyman, G H, Papahadjopoulos, D & Preisler, H D, Biochim biophys acta 448 (1976) 460. 23. Harris, H L & Zalik, S E, J cell physiol 83 (1974) 359. 24. - Differentiation 7 (1976) 83. 25. Cook, G M W, Zalik, S E, Miles, N & Scott, V. III Int conf on differentiation, p. 32a. Minneapolis, Minn (1978).

Received March 30, 1978 Revised version received June 12, 1978 Accepted June 15, 1978