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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 con- canavalin 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 label- ing. 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 re- sulted 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 ob- served 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% paraform- aldehyde (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).
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