Effects of Colchicine, Cytochalasin B

EFFECTS OF COLCHICINE, CYTOCHALASIN B,

AND ~-DEOXYGLUCOSE ON THE TOPOGRAPHICAL

ORGANIZATION OF SURFACE-BOUND CONCANAVALIN A

IN NORMAL AND TRANSFORMED FIBROBLASTS

THOMAS E. UKENA, JOAN Z. BORYSENKO, MORRIS J. KARNOVSKY, and RICHARD D. BERLIN

From the Departments of Physiology and Pathology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT The distribution of surface-bound concanavalin A on the membranes of 3T3, and simian virus 40-transformed 3T3 cultured mouse fibroblasts was examined using a shadow-cast replica technique with a hemocyanin marker. When cells were prefixed in paraformaldebydc, the binding site distribution was always random on both cell types. On the other hand, labeling of transformed cells with concanavalin A (Con A) and hemocyanin at 37°C resulted in the organization of Con A binding sites (CABS) into clusters (primary organization) which were not present on the pseudopodia and other peripheral areas of the membrane (secondary organization). Treat- ment of transformed cells with colchicine, cytochalasin B, or 2-deoxyglucose did not alter the inherent random distribution of binding sites as determined by fixation before labeling. However, these drugs produced marked changes in the secondary (but not the primary) organization of CABS on transformed cells labeled at 37°C. Colchicine treatment resulted in the formation of a caplike aggregation of binding site clusters near the center of the cell, whereas cytochalasin B and 2-deoxyglucose led to the formation of patches of CABS over the entire membrane, eliminating the inward displacement of patches observed on untreated cells. The distribution of bound Con A on normal cells (3T3) at 37°C was always random, in both control and drug4reated preparations. Pretreatment of cells with Con A enhanced the effect of colchicine on cell morphology, but inhibited the morphological effects of cytochalasin B. The mechanisms that determine receptor movement and disposition are discussed.

INTRODUCTION Many physiological changes which arise when transformation include changes in contact inhibi- cells are transformed from a normal to a malignant tion of growth (1), cell shape (2), membrane trans- state can be ascribed to alterations of membrane port (3-5), and surface-related phenomena such function. Thus, physiological manifestations of as agglutination by plant lectins (6-I 1). Trans-

70 THE JOURNAL OF CELL BIOLOOY • VOLUME 6], 1974 • pages 70-82 formation is usually accompanied by increased We report here alterations of the secondary agglutinability ofceUs by the lectirt concanavalin A organization of CABS effected by colchicine, (Con A) 1 (9). Although it was originally hypothe- 2-deoxyglucose, and cytochalasin B which permit, sized that the greater agglutinability of trans- to some extent, assessment of the contribution of formed ceils might be associated with increased primary as opposed to secondary organization to numbers or unmasking of "cryptic" Con A bind- the physiological properties of cells treated with ing sites (CABS) (8, 12), the bulk of the available Con A. These experiments also provide evidence evidence suggests that this is not the case (13-16). that the secondary organization of surface-bound The relative agglutinability of cells by Con A Con A is modified by colchicine and other drugs and other plant lectins has, however, been cor- that depolymerize microtubules and suggest related with the topographical (surface) distribu- possible mechanisms involved in the movements tion of bound Con A (17). Thus the CABS ~ on of CABS across the cell surface. normal ("contact inhibited") fibroblasts are randomly dispersed, while virus-transformed cells MATERIALS AND METHODS have CABS gathered into clusters (17, 18). The 3T3, and 3T3 cells transformed by SV40 virus density of CABS in these clusters apparently (SV3T3) (generous gifts from Dr. Howard Green of favors cross-linking of adjacent cells by the tetra- the Massachusetts Institute of Technology, Cam- valent lectin molecule, leading to cell agglutina- bridge, Mass.), were grown to confluency in plastic tion. Recently it has been shown that the cluster- tissue culture flasks (Falcon Plastics, Division of Bio- ing of CABS on transformed cells is induced by Quest, Oxnard, Calif.). Cultures were incubated in the multivalent Con A itself, whereas the inherent 10% CO2 in air. The culture medium was Dulbecco's binding site distribution on the cell surface is modified Eagle's medium (DMEM) (Grand Island Biological Co., Grand Island, N. Y.) supplemented dispersed and random (18, 19). The ability of with 10% heat-inactivated (56°C, 1 h) calf serum Con A to induce clustering of its binding site~ is and 10% tryptose phosphate broth (Microbiological one characteristic of the transformed state. Associates, Inc., Bethesda, hid.), and containing 75 In the course of our experiments we have ob- U/ml penicillin and 75 U/ml streptomycin. Cul- served that the CABS clusters themselves assume a tures were examined periodically for mycoplasma characteristic distribution on the surface of the contamination. Cells were removed from culture cell membrane, since they are not usually ob- flasks with 0.25% trypsin, plated onto 15-ram glass served in the distal areas of the pseudopodia or on cover slips contained in 35- )< 10-ram Petri dishes, the peripheral area of the cell membrane. Thus and allowed to grow for 36 h before use in any ex- two types of organization contribute to the in- periment. The ceils were plated at low density, so that there was little cell-to-cell contact on the mono- duced topography of CABS on the SV3T3 cell layers. membrane. We define a primary organization Solutions of colchicine (10 -6 M, Sigma Chemical which corresponds to the clustering of CABS, and Co., St. Louis, Mo.), vinblastine sulfate (10 -6 M, a secondary organization which refers to the Eli Lilly and Co., Indianapolis, Ind.), Colcemid spatial distribution of the clusters on the cell (10-6 M, Ciba Pharmaceutical Co., Summit, N. J.), membrane. In transformed cells this secondary 2-deoxyglucose (10 raM, Sigma Chemical Co.), and organization of CABS is stringently correlated alpha-metbyl-n-mannoside (50 raM, Pfanstiehl Labs., with the clustering phenomenon, hence its rela- Inc., Waukegan, Ill.), were prepared in DMEM tive contribution to physiologic properties is still buffered with Tricine (Sigma Chemical Co.) to pH uncertain. 7.4. Lumicolchicine was prepared as described by Wilson and Friedkin (20). Cytochalasin B (ICI Re- 1 Abbreviations used in this paper: CABS, concanavalin search Laboratories, Cheshire, England), (1 mg/ml), A binding sites; Con A, concanavalin A; Con A/H, was dissolved as a stock solution in dimethyl sulfoxide sequential treatment of cells with Con A and hemo- and diluted to 10/zg/ml in DMEM for use in experi- cyanin; DMEM, Dulbecco's modified Eagle's ments. In control experiments it was shown that the medium; PBS, Dulbecco's phosphate-buffered saline; low (1.0%) concentrations of dimethyl sulfoxide PFA, paraformaldehyde; SV3T3, 3T3 cells trans- present in the final dilution had no apparent effect formed by SV40 virus. on the cells. 2 Since these surface elements are observed only after To mark the surface distribution of Con A, we they have bound Con A, it is to be understood that employed the technique of Smith and Revel (21), CABS as used here refers to the Con A receptor using hemocyanin (Busycon canaliculatum) which binds complex on the cell surface. to Con A molecules and is readily visible in conven-

UKENA E~ AL. Topography of Concanavalin A Binding 71 tional shadow-cast replicas. The details of this tech- PFA before Con A/H labeling, or when labeling is nique have been described previously (18). Solutions performed entirely at 4°C, is random (18). When of Con A (twice crystallized, Miles-Yeda Ltd., Miles clustering of CABS is induced by Con A, hemocy- Laboratories, Inc., Elkhart, Ind.), and hemocyanin anin clusters cover much of the cell surface, but were prepared in Dulbecco's phosphate-buffered are absent from the distal aspects of pseudopodia saline (PBS) at pH 7.4. In most experiments the cover slips were first in- and usually from the edges of the cells as welP cubated for 60 rain in DMEM with Trieine, with or (Fig. 1). To test whether this localization of the without drugs. After three serial rinses in PBS patches represented CABS in transit to another (20°C), the cover slips were then incubated in Con distribution, or whether it constituted the final A (100 #g/ml at 37°C or 200 #g/ml at 4°C) for 10 distribution, we incubated the cells in DMEM mAn. The cover slips were again rinsed serially three for various periods of time after Con A/H treat- times and incubated a further 10 mAn in hemocyanin ment (before fixation). After 60 min of incuba- (500 #g/ml at 37°C or 1.0 mg/ml at 4°C). In ex- tion in DMEM, there was no change in the pattern periments where cells were treated with drugs, the of CABS relative to controls (labeled and fixed same compound was present in the labeling solutions. immediately). Observation of the cells by phase In some cases the cells were fixed in 1% paraformaldehyde (PFA) (MC & B Manufacturing Chem- microscopy showed that over a 20-min period at ists, Norwood, Ohio) in PBS (pH 7.4) for 10 man at 37°C, with or without Con A, there was no 37°C after the drug treatment. In one type of experi- noticeable movement of the cells nor any change ment the cells were first labeled with Con A and in the shape of the pseudopodia. Since exposure of hemocyanin (Con A/H) and then incubated for 60 lymphocytes to low temperatures has been shown rain in DMEM with or without the addition of drugs. to affect the final distribution of CABS on lympho- Cells were fixed and prepared for electron micros- cytes (25), perhaps by causing depolymerization of copy as described previously (18). Replicas were microtubular protein (26), we studied CABS dis- examined in an AEI EM 801 electron microscope tribution as a function of temperature. When cells with an accelerating voltage of 60-80 kV. were treated with Con A and hemocyanin at [x25I]Con A was prepared by the method of Helm- kamp et al. (22) and purified as described previously 4°C and then warmed to 37°C for 10 or 60 rain, (18). Binding of [12~I]Con A was studied using the the distribution of CABS was identical to that of same conditions and concentrations employed in the ceils labeled at 37°C (never having beeen cooled). morphological studies. Cover slips treated with 3T3 : 3T3 ceils treated with Con A/H with or [125I]Con A were rinsed in PBS and the attached without prior fixation show hemocyanin dis- cells were dissolved in Lowry's solution A (23). tributed in a random pattern over the entire cell Samples were taken for determination of protein membrane, including pseudopodia (18). (Lowry et al. [23]) and radioactivity by liquid scintillation. ATP was extracted from the cells in 5% per- Effect of Colchieine on the Distribution ehloric acid at 4°C. The extract was neutralized and of CABS ATP was measured employing a luciferin-luciferase preparation (Sigma), using the method of Addanki SV3T3: Colchicine (10 -6 M, 60 min) had a et al. (24). striking morphological effect on the ceils. They became rounded-up, and numerous long, stiff RESULTS microspikes originated from a region of membrane replete with filopodia near the center of the cell Distribution of CABS on Normal and (presumably the nuclear region). These micro- Transformed Mouse Fibroblasts spikes protrude the plasma membrane and extend to the cell borders. When cells were treated SV3T3: Monolayer cultures of SV3T3 ceils with colchicine and fixed in 1% PFA before Con contain a pleomorphic population of both rounded A/H treatment, the hemocyanin was dispersed. and fibroblast-like cells showing surface filopodia Thus colchicine did not change the inherent dis- over much of the cell surface, but particularly tribution of CABS. Monolayers that were pre- concentrated over the central area of the cell, pretreated with colchicine and labeled with Con A/H sumably the nuclear region. Large pseudopodia project from the lateral cell borders. As reported 3 We have recently observed a similar distribution of previously, the inherent distribution of CABS on Con A/H on spontaneously transformed baby hamster these cells, seen when the cells are fixed in 1% fibroblasts.

72 ThE JOURNAL OF CELL BIOLOGY " VOLUME 61, 1974 FIG. 1 Shadow-cast replica of an SV3T3 cell surface showing clusters of hemocyanin (arrows) after Con A/H treatment at 37°C. Clusters are absent from edges of cell (E) and pseudopodia (P). Direction of shadow is from bottom to top in all replicas. Bar = 1/zm. X 9,500.

UKENA ET AL. Topography of Coneanavalin A Binding 73 FI~. ~ SV3T3 cell treated with 10-6 M colchicine before Con A/H labeling. Mierospikes (M) extend from the central area of the cell (C) past the cell edges (E). Large aggregates of hemoeyanin (arrows) are seen in the center of the cell, and small clumps (circles) are often associated with the points of origin of the mierospikes. Bar = 1 #m. X 13,000.

74 Tax JOURNAL OF CELL BIOLOGY • VOLI~E 61, 1974 in the presence of the alkaloid showed no change in the primary organization of CABS (cluster formation), but the distribution of the clusters over the surface of the cell (secondary organization) was very different from that in control preparations (Fig. 2). Patches were found pre- dominantly in the central area of the cell, often in large aggregates, and, in addition, often at the points of origin of the microspikes. Clusters were also preferentially associated with areas of pre- sumed membrane activity such as filopodia. Over- all, the CABS had moved centripetally, but the persistence of independent clusters distinguishes their organization from the tight "caps" of receptors formed on lymphocytes under similar conditions (27). Thus colchicine did not change the inherent distribution of CABS nor their primary organization (patches) but rather the ordering of patches on the membrane which we refer to as a second level of CABS organization. Identical effects were seen when monolayers were treated with vinblastine sulfate (10 -6 M), or Colcemid (10 -8 M). However, SV3T3 cells treated with lumicolchicine (10 -6 M), a photo- chemical derivative of colchicine that does not bind to microtubular proteins (28), showed normal morphology and the control distribution FIGURE8 (a) Thin section of Con A/H-treated SVgT8 of hemocyanin patches. cell revealing a network of microfilaments (]If) and Thin sections of SV3T3 cells revealed a net- microtubules (Mt) subjacent to the plasma membrane. work of microfilaments and microtubules subjacent Hemocyanin (arrow) is present on the cell surface. to the plasma membrane (Fig. 3 a). Treatment of >( 75,000. (b) SV3T3 cell treated with 10-6 M vin- these cells with 10-6 M vinblastine for 60 min at blastine before Con A/H labeling. Microtubulcs are 37°C resulted in the disappearance of most of the absent. Bar = 0.25 pm. X 75,000. microtubules, while the microfilament network re- mained apparently intact (Fig. 3 b). In addition, affect the state of aggregation of the Con A sub- profiles of the microspikes revealed them to be units or Con A-saccharide interactions (25). straight tubular projections of membrane above 3r3 : The morphological effects of colchicine, the cell surface which were filled with microfila- vinblastine, and Colcemid in these cells were ments. similar to those seen with SV3T3 cells. However, Colchicine did not affect the time-course or the hemocyanin was present in a random, dis- kinetics of binding of [l~sI]Con A to SV3T3 or persed pattern, indistinguishable from control 3T3 cells as determined on monolayers. The de- monolayers. sign of the labeling experiments did not permit comparison of the relative amounts of Con A Effect of Cytochalasin B on the Distribution bound to normal vs. transformed cells. The same of CABS amount (90%) of bound [l~sI]Con A was removed SV3T3: Incubation of SV3T3 cells in cyto- from control and colchieine-treated monolayers by chalasin B (10 ttg/ml) led to changes in the mor- alpha-methyl-D-mannoside (50 raM), indicating phological appearance of the cells that were con- that the alkaloid had neither induced nor de- sistent with previous observations (29). Long pressed internalization of bound Con A, which pseudopodia terminating in stellate arborizations does not occur to any great extent with these cells radiated from severely contracted cell bodies. under any conditions. Also, colchicine does not In contrast to the centripetal distribution of

U~E~A EV AL. Topographyof Concanavalin A Binding 75 FIG. 4 SV3T3 cell treated with 10 btg/ml cytochalasin B before Con A/H labeling. Long pseudopodia (P) terminate in stellate arborizations (A) which radiate from a contracted cell body (CB). Clusters of hemocyanin (circles) are present near cell edges as well as within pseudopodia and arborizations. Bar = 1 #m. X 9,500.

76 THE JOURNAL OF CELL BIOLOGY • VOLUME 61, 1974 patches on control cells, cytochalasin B-treated of colchicine on cell morphology. Con A/H- cells showed patches distributed over the entire treated cells were incubated for various periods cell surface including the long, stellate pseudo- in colchicine (10 -6 M) and compared with con- podia (Fig. 4). This effect was readily reversible. trois (PBS, colchicine). Pretreatment with Con When cytochalasin-treated monolayers were A/H or Con A alone greatly accelerated the washed and incubated in DMEM for 60 min rounding-up of the cells caused by colchicine. before Con A/H treatment, the morphology and Although a sufficiently long incubation (120 hemocyanin pattern was the same as in control min) in colchicine resulted in rounding-up of SV3T3 monolayers. When cytochalasin B- virtually all the cells, Con A pretreated or not, treated cells were fixed before Con A/H labeling, the cells not pretreated with Con A took con- the hemocyanin pattern was random. Cytochala- siderably longer to reach the final characteristic sin has been shown to inhibit the membrane colchicine-induced morphology. At 60 min only transport of various substances such as glucose 25 % of the control cells were completely rounded- (30-33). To test the possibility that the cyto- up, while 100 % of the Con A-treated cells were chalasin effect was mediated indirectly through rounded. This effect was related to the dose of inhibition of transport, monolayers were treated Con A employed; 5 #g/ml had little effect and with cytochalasin in PBS (which contains no 20 ~g/ml had an intermediate effect. When the nonelectrolytes) instead of DMEM. The results experiment was repeated with I0-~ M colchicine, of this experiment were identical with the pre- the morphological changes occurred more quickly vious one, for both control and cytochalasin- in both control and Con A-treated cells, but the treated cells. Furthermore, an 80-rain incubation Con A-treated cells still rounded-up more rapidly in cytochalasin B caused no change in the intra- than PBS controls. The final binding site dis- cellular levels of ATP, as previously reported tribution of Con A/H-colchicine-treated cells (34). was similar to that seen on cells pretreated with 3w3: Cytochalasin B produced the same colcbicine. changes in morphology seen in SV3T3 cells, CYTOCHALASIN B : In contrast, treatment of without any effect on the distribution of hemo- cells with Con A or Con A/H before incubation cyanin on Con A/H-treated 3T3 cells. in cytochalasin B prevented much of the rounding- up of the cell body that is usually observed in Effect of 2-Deoxyglucose on cells treated with cytochalasin B. This inhibitory effect was also Con A dose dependent. The CABS Distribution CABS distribution on Con A-cytochalasin B- To test the requirement for intracellular energy treated cells was similar to that of control (Con stores in determining the organization of CABS A only) cells. Thus cytochalasin B does not cause on the cell surface, we treated SV3T3 cells with the patches to move back to the peripheral areas l0 mM 2-deoxyglucose for 60 rain before Con of the membrane after they have migrated cen- A/H. This procedure depleted the levels of ATP trally as a result of prior Con A treatment. by 80%. The morphological appearance of 2- DISCUSSION deoxyglucose-treated ceils was very similar to that of cytochalasin-treated ceUs, and, as with Colchicine, cytochalasin B, and 2-deoxyglucose cytochalasin-treated cells, the hemocyanin marker have been used to investigate the mechanisms was found in patches distributed over the entire involved in determining the surface topography cell surface. When 2-deoxyglucose-treated ceils of receptor-Con A complexes on normal and were fixed before Con A/H labeling, the hemo- transformed fibroblasts. We have shown that: (a) cyanin pattern was random. none of the drugs affected the inherent random distribution of CABS; (b) none of the drugs af- Colchicine and Cytochalasin after Con A /H fected the capacity of Con A to induce clustering of CABS (primary organization) on SV3T3 cells. COLGHICINE: Con A/H pretreatment did However, all three drugs affected the topo- not noticeably alter the morphology of SV3T3 graphical distribution of the induced clusters on cells incubated for 60 rain in DMEM buffered transformed cells (secondary organization). The with Tricine. However, pretreatment of SV3T3 results of these experiments are summarized in cells with Con A or Con A/H enhances the effect Table I.

UKE~A ET AL, Topographyof Concanavalin A Binding 77 TABLE I their general shape during the Con A/H labeling Distribution of CABS on Fibroblasts at 37°C period. Therefore, the observed secondary CABS after Con A/H and Drug Treatment* topography must result from a redistribution of CABS on the cell surface that is not related to Drug SV3T3 Cells 3T3 Cells gross cell movement. In this regard Ryan et al. None Clusters, with- Dispersed have recently shown that cell movement does drawn from not affect the formation of a single compact periphery aggregate of bound Con A (capping) on human None-prefix Dispersed Dispersed polymorphonuclear leukocytes, but rather the Colehieine Central cap Dispersed final location of the capped Con A (39). Caps Colehieine- Dispersed Dispersed were found at the trailing edge of actively moving prefix cells, but over the central area of nonmotile cells Cytochalasin B Random clusters Dispersed (39), the latter in a fashion similar to that ob- Cytoehalasin Dispersed Dispersed served on colchicine-treated SV3T3 cells shown B-prefix 2-Deoxyglucose Random clusters Dispersed here. 2-Deoxyglucose- Dispersed Dispersed Mouse fibroblasts treated with antibodies to prefix surface H-2 antigens showed a labeling pattern similar to the CABS topography on SV3T3 ceils * Cells were treated with drugs and labeled with at 37°C (40). This organization of surface com- Con A/H in the presence of the same drug, as de- ponents was also prevented by metabolic in- scribed in the text. In "prefix" experiments cells hibitors and low temperature, but, in contrast to were treated with drugs and fixed in 1% para- our results with CABS, Colcemid treatment did formaldehyde before ConA/H labeling. not lead to cap formation in this system (40). The translocation of CABS in control and al- The manner by which CABS patches reach their final distribution on the membrane is un- kaloid-treated SV3T3 cells may represent inde- known, and is obviously a complex process. pendent movement of CABS across the cell sur- Data from several laboratories have suggested that face, perhaps accompanied by further cross- linking of CABS, or, alternatively, it could be the distribution and mobility of surface receptors caused by a bulk membrane flow (41) that carries may be controlled by cytoplasmic structures. Ber- CABS along. In the former case, the movement lin and Ukena (35-37) and more recently Edel- could be simply the result of diffusion, influenced man et al. (25) have proposed that the mobility by the mobility of CABS, and the size and density of cell surface receptors is regulated by a colchi- of clumps, as well as membrane activity such as cine-sensitive assembly somewhere in the vicinity of the plasma membrane• The distribution of "ruffling" or extension and retraction of filopodia. receptors, therefore, is presumably related to the Of relevance to this latter possibility are the microcinematographic studies of Vasiliev et al. state of a "colchicine-binding protein" which may correspond to the microtubule system of the showing that Colcemid and vinblastine induce movements of normally stable areas of fibroblast cell (38). cell membranes (42). In colchicine-treated SV3T3 The effects of colchicine on the secondary organization of CABS in our system are consistent cells, clumps were often found at the centrally located bases of the microspikes, suggesting that with, but do not constitute proof of, a role for microtubules in regulating mobility of surface membrane activity influences their distribution. receptors. In addition, variables such as cell motility, the microfilament network, local mem- Relation of Primary to brane movements, and the state of fluidity of the Secondary Organization membrane per se must be considered in any model Our results do not define whether cluster forma- of receptor disposition, yet their respective roles tion and migration of CABS occur separately or have not been fully clarified at present. simultaneously. However, treatment of SV3T3 Diffusion and Membrane "Activity" as cells with cytochalasin B or 2-deoxyglucose pre- vents the usual migration of CABS clusters away Determinants of CABS Topography from the cell periphery and out of pseudopodia, SV3T3 cells observed by phase-contrast showing that CABS can be clustered at any point microscopy do not appear to move or change on the membrane. These data suggest that cluster

78 THE JOUI~NAL OF CELL BIOLOGY • VOLUME 61, 1974 formation can be accounted for by local diffusion secondary topography is attained. Yahara and and the cross-linking properties of the multivalent Edelman (44) have recently reported that Con A Con A molecule. On the other hand, centripetal in doses greater than 5 #g/ml inhibits the forma- migration of CABS has not been observed in the tion of caps on lymphocytes by Con A itself and absence of patch formation. For example, the also by antibodies directed against surface im- ligand receptor complex on the 3T3 cell mem- munoglobulins. The stable secondary topography brane is always dispersed and distributed over the of CABS on SV3T3 cells may represent a similar entire membrane, even after colchicine treatment. inhibition of receptor mobility. Although Con Thus, cluster formation might trigger or facilitate A does not inhibit the initial movements of CABS centripetal movement of CABS, or at least be in- on SV3T3 cells, the clusters may reach a limiting dicative of a membrane state which allows this size or density as they move in from the edges movement of CABS. of the cells, resulting in restriction of their mobility, perhaps due to increased inertia of larger Energy Requirement for CABS Movement - clumps or to induced changes in structures regu- Possible Role of Mierofilaments lating receptor mobility, as proposed by Edelman et al. (25). The action of 2-deoxyglucose in causing re- Our observations of the effects of colchicine traction of the SV3T3 cell body and preventing and cytochalasin B on cells pretreated with Con the secondary topographical redistribution of A also suggest that cross-linking of membrane CABS indicates an energy requirement for the proteins by Con A has significant effects on mem- centripetal movement of CABS. The similar brane properties. Both of these compounds cause effect produced by cytochalasin B suggests that ceils to condense into relatively spherical forms. this energy requirement may be mediated through When the CABS clusters remain dispersed over the microfilament system (29). However, in much of the membrane surface, as on cells treated preliminary experiments we were unable to ob- with Con A/H and then cytochalasin B, the serve a morphological effect of cytochalasin B rounding-up of the cells is greatly inhibited. In on the network of microfilaments closely apposed the Con A-colchicine-treated cells where CABS to the membrane. Although the effect of cyto- clusters aggregate in a small central area of the chalasin B on CABS was shown to be unrelated to membrane, rounding-up occurs much more membrane transport, it is, of course, possible that rapidly than in cells treated with colchicine this action represents another direct effect on alone. Thus, cross-linking of CABS on the periph- the membrane, a conclusion that is supported eral areas of the membrane appears to stabilize the by the rapid reversal of the cytochalasin B effect. cell against the morphological effects of cyto- Metabolic inhibitors have also been shown to chalasin B and redistribution of these receptors inhibit cap formation in lymphocytes (43) and into a small area of the cell surface accelerates polymorphonuclear leukocytes (39). These ob- the rounding-up induced by colchicine. servations can be taken to favor the involvement The temperature at which the cells are treated of membrane flow or active translational forces with Con A has no effect on the final distribution on CABS clumps, but 2-deoxyglucose and cyto- of hemocyanin after the ceils have been warmed ehalasin B may influence other membrane prop- to 37°C. This result is in contrast to recent evi- erties. dence (25) showing that treatment of lympho- Restricted Mobility of the Con A cytes with Con A at 4°C followed by warming Receptor Complex resulted in cap formation, a result attributed to slow reassembly of cold-sensitive microtubular When cells were treated with Con A/H in the protein. The discrepancy may be attributed to cold, rinsed, and then warmed to 37°C, the sec- the different types of cells used in the two experi- ondary organization of CABS was attained in 10 ments, or could imply the involvement of other rain (the shortest time tested). This topography factors in determining the final CABS topography. was stable over at least a 1-h period. Thus the interaction of Con A with its surface receptors is Mechanism of Colchicine Effect on followed rapidly by formation of CABS clusters CABS Topography and movement of CABS toward the center of the cell. However, the process of aggregation and When SV3T3 cells are treated with colchicine translation slows or stops when the characteristic or related alkaloids, the CABS move to the center

UKENA ET AL Topographyof Concanavalln A Binding 79 of the cell forming an aggregate of clusters simi- In summary, two degrees of binding site orga- lar to a cap. Similarly, Edelman et al. report that nization have been described: (a) the rearrange- colchicine antagonizes the inhibition of lympho- ment of CABS into clusters (primary), and (b) cyte cap formation by Con A (25). The pharma- the ordering of these clusters over the cell surface cology of this effect parallels the actions of these (secondary). Both types of binding site organiza- drugs on microtubular protein, since (a) colchi- tion must be considered in any analysis of the cine is effective at low doses (10 -6 M), (b) vin- properties of Con A-treated cells. Pharmacologi- blastine sulfate and Colcemid have identical cal evidence suggests that microfilaments and effects, and (c) lumicolchicine has no effect on microtubular proteins may influence CABS the topography of CABS. These actions of col- distribution on the SV3T3 cell surface. chicine may involve nonmembrane structures such as subsurface microtubules, which are shown to This research was supported by grants GM-01235 be absent after the addition of vinblastine or, and H E-01925 from the National Institutes of Health, alternatively, colchicine may have direct actions United States Public Health Service, grant 1 RO1 on drug-sensitive structures within the cell mem- CA L5544-01 from the National Cancer Institute, brane. Our results do not distinguish between and grant 7 ROI AM17415-01 from the National Institute of Arthritis, Metabolism, and Digestive these alternatives, but in any case, the result of Diseases. colchicine treatment is to allow or promote free Received for publication I September 1973, and in revised movement of CABS, leading to their aggregation form 6 December 1973. and redistribution.

Agglutination by Con A of SV3T3 Cells REFERENCES

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82 THE JOURNAL OF CELL BXOLOGY • VOLUME 61, 1974