Proc. Nat. Acad. Sci. USA Vol. 72, No. 2, pp. 572-576, February 1975
Distribution of Membrane Particles and Gap Junctions in Normal and Transformed 3T3 Cells Studied In Situ, in Suspension, and Treated with Concanavalin A (freeze-fracture/cancer/electron microscopy/viral transformation/cell culture)
PEDRO PINTO DA SILVA* AND ADOLFO MARTINEZ-PALOMOt * The Salk Institute for Biological Studies, San Diego, California 92112; and t Centro de Investigacion del I.P.N., Mexico 14, D.F., Mexico
Communicated by Robert W. Holley, November 29, 1974 Freeze-fracture techniques were used to AB STRACT study the ultrastructural distribution of plasma membrane particles in cultures of normal Balb/c and Swiss 3T3, and simian virus 40- or murine sarcoma virus-transformed fibroblasts. No apparent differences were observed. Cultures fixed in situ show a seemingly random distribution of membrane particles both in normal or in transformed cells. Treatment of cell cultures in situ with concanavalin A does not result in an altered pattern of particle distribution. EDTA-induced suspension of normal or transformed cells does not result, per se, in modification of the type of membrane particle distribution seen in cells fixed in situ. However, upon further incubation, a proportion of normal or transformed cells in suspension show a varying degree of particle aggregation following a network pattern. Concanavalin A treatment of normal and transformed cells in suspension does not result in a specific change of the pattern of particle distribution. Because it has been established that treatment with concanavalin A of simian virus 40-transformed Balb/c 3T3 fibroblasts causes pronounced clustering of the concanavalin A receptors at the outer surface, our results probably imply independence of membrane particles and concanavalin A receptors of these transformed cells.
receptors may also occur in normal Balb/c 3T3 fibroblasts
(7).
Unlike conventional thin-section electron microscope studies, freeze-fracture techniques allow further observation of the inner structure of biological membranes. During fracture the frozen membrane is split along the central plane of the bilayer, creating faces that contain numerous particles (8). Studies of the chemistry and topology of the particles have been mostly restricted to isolated erythrocyte membranes. In these membranes, combination of freeze-fracture and freeze-etch (sublimation) techniques has shown that the particles represent protein-containing structures that are intercalated and traverse the bilayer domain and expose antigens, lectin, and viral receptors and acidic sites at the outer surface (9-12). It is unlikely, however, that conclusions derived from the study of isolated erythrocyte membranes can be freely extrapolated as a model for the plasma membranes of live, eukaryotic cells. We have recently shown that on the plasma membranes of Entamoeba histolytica trophozoites, surface receptors may not be associated with the particles, since particles and surface receptors may move independently (13, 14). Independence of particles and Con A receptors has also been suggested in lymphocyte plasma membranes (15). This means that fluidity of the membrane can be considered at two separate levels: peripheral membrane fluidity (displayed by peripheral membrane components) and integral membrane fluidity (displayed by integral membrane components, such as those represented by the membrane particles). In the erythrocyte membrane, these concepts intersect because the membrane particles represent integral membrane components that are expressed as antigens and receptors at the outer membrane surface and, in consequence, movement of the particles implies movement of surface receptors
In recent years much attention has been devoted to the study of the composition, structure, and fluidity of the plasma membranes of normal and transformed cells. The use of these cell systems as experimental models of normal and cancerous growth stems, to a large extent, from the fact that while cultures of normal cells exhibit density-dependent inhibition of growth, in cultures of transformed cells proliferation continues after confluency (1). In initial studies of the surface distribution of concanavalin A (Con A) receptors in normal and transformed cells, it was concluded that the inherent distribution of these receptors was random. in normal cells while it was clustered or discontinuous in transformed cells (2-4). In these studies the membranes were either insufficiently fixed or not fixed before treatment with the lectin, and it was later shown that while the native distribution of Con A sites is random in both normal and transformed Balb/c 3T3 fibroblasts, treatment with Con A induces clustering of the receptors on the transformed cells (5, 6). Recently it has been reported that clustering of Con A
(9-12).
Freeze-fracture observations of the plasma membranes of secondary explant fibroblasts and of fibroblasts transformed by Rous sarcoma virus revealed random distribution in normal and transformed cells, as no clustering of particles was observed except for that which corresponded to gap junctions between normal or transformed cells (16). Recently, however, it was reported that in normal 3T3 fibroblasts (but not in cells transformed by simian virus 40) an intensely clustered distribution of the plasma membrane particles occurred upon the establishment of cell contacts, the incidence of which peaked as the cultures became confluent (17). Clustering of membrane particles was also reported in murine plasmacytoma cells treated with Con A and thought to represent,
Abbreviations: Con A, concanavalin A; 3T3 Balb, Balb/c 3T3 clone A31 fibroblasts; 3T3 Swiss, Swiss 3T3 clone A4 fibroblasts; SV- or MSV-Balb, Balb/c 3T3 clone A31 fibroblasts transformed by simian virus 40 or murine sarcoma virus, respectively; SVSwiss, Swiss 3T3 clone A4 fibroblasts transformed by simian virus 40.
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FIGS. 1-3. Fracture faces of the plasma membranes of 3T3 Balb (Fig. 1) and SV-Balb (Figs. 2 and 3) cells fixed in situ. With the exception of gap junctions (unusually numerous in Fig. 1), the particles are randomly distributed. Two loose particle clusters (Fig. 1, arrows) may represent stages of junction formation or disintegration. Gap junctions may appear in groups (Fig. 1) or isolated (Fig. 3, arrow). Figs. 1 and 3, X55,000; Fig. 2, X150,000.
in these cells, an acquired feature of the plasma membrane accompanying Con A-induced agglutination (18). In this study we have used Balb/c and Swiss 3T3 fibroblasts, normal and transformed by simian virus 40 (SV 40)
and murine sarcoma virus (\ISV), and report the absence of differences in the native pattern of distribution of the membrane particles as well as the absence of alteration in the pattern of distribution of membrane particles of normal and
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FIGs. 4 and 5. Plasma membrane fracture face A of 3T3 Balb (Fig. 4) and SV-Balb (Fig. 5) cells treated in situ with Con A. The pattern of particle distribution is not altered. X60,000. transformed cells treated with Con A. Gap junctions identified in cultures of normal or of transformed cells.
are
MATERIALS AND METHODS
Cells. Untransformed and SV40- or MSV-transformed Balb/c 3T3 A31, and untransformed Swiss 3T3 A4 and SV40transformed cells were used. The cells were plated at densities of 1 to 3 X 103 cells per cm2 in 75-cm2 Falcon flasks and were confluent within 3-5 days. Saturation densities of untransformed cells were 3 to 5 X 104 cells per cm2. The cells were grown in Dulbecco's modified Eigle's medium supplemented with 10% (v/v) calf serum (heat-inactivated for Balb/c cultures) changed at 3-day intervals. Treatment. (a) Cells in situ: Cell cultures were fixed in situ by addition of 50% glutaraldehyde to the medium to a final concentration of 1.5% and incubated for 30 min at 370. In experiments designed to study the effect of Con A, cell cultures were washed three times in phosphate-buffered saline (pH 7.3) and then incubated with 10 ml of Con A (10 and 100 ,ug/ml) in phosphate-buffered saline for 30 min at 37°. Controls were incubated in phosphate-buffered saline without Con A for the same period. The cells were then fixed as above. (b) Cells in suspension: Cell suspensions were obtained by incubation in 2 mM EDTA in Ca2+, Mg2+-free phosphatebuffered saline for 30 min at 370 (18). The cells were either fixed immediately in 1.5% glutaraldehyde for 30 min at 37° or washed twice and incubated for 30 min at 370 in the presence or absence. of Con A (10 or 100 ,ug/ml). The cells were then fixed as at ove before glycerol impregnation. (c) Glycerol impregnation: Because glycerol impregnation of unfixed cells can cause alterations of the pattern of distribution of plasma membrane particles (13, 19), effective glutaraldehyde fixation is necessary before glycerol impregnation. After glutaraldehyde fixation, cells fixed were removed from the dish with a Teflon spatula. All cells were suspended in about 1 ml of phosphate-buffered saline, gradually impregnated by dropwise addition of 25% glycerol in phosphatebuffered saline at 370 over a period of 10 min, incubated for 30 min at 370, and cooled for 1 hr at room temperature. To test the effect of glycerol on unfixed cells in suspension, some
cell cultures were also incubated in glycerol-phosphate-buffered saline without glutaraldehyde prefixation. Freeze-Fracture. The cells were frozen in the liquid phase of partially solidified Freon 22 and freeze-fractured in Balzer's 501 device equipped with an electron gun for platinum-carbon evaporation. The replicas were recovered, cleaned, and observed with an Hitachi HU-12 electron microscope at 75 kV. The micrographs are mounted with shadow direction from the bottom; shadows are white. Only fracture faces of the plasma membranes are shown. The membranes are split during fracture, and A face represents the outer aspect of the inner half of the bilayered regions of the membrane plus associated particles that protrude into the outer half of the membrane (8); vice versa for B face. The word "random" is used here not in the mathematical sense but, arbitrarily, to describe that the distribution of membrane particles does not follow a readily recognizable pattern. RESULTS
Cells In Situ. Observation of the fracture faces of plasma membranes of 3T3 Balb, 3T3 Swiss, SV-Balb, SV-Swiss, and MSV-Balb cells from cultures fined with 1.5% glutaraldehyde for 30 min at 370 directly in the culture dish invariably revealed a random distribution of membrane particles (Figs.1 and 3). As seen in freeze-fractured plasma membranes of other eukaryotic cells, there is remarkable heterogeneity in the size of the membrane particles, as all transitions can be observed from a definite particle to a clear smooth region. Smaller rugosities ("subparticles," ref. 13) were best visualized over membrane regions that were exposed to a lower angle of shadow. Because a previous report proposed that clustering of the particles of 3T3 Balb progressively increased with the establishment of cell contacts (17), we observed these cells in subconfluent (about 70% occupation of substrate) and confluent states. Also, because of the long periods of culture required to reach cell confluency and maximal incidence of particle clustering (Fig. 2 of ref. 17), we also observed 15-dayold cell cultures, i.e., cells that remained in culture for 10 days after reaching confluency. In all cases the distribution of particles was random. The only particle clusters found could
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FIGS. 6-11. Patterns of particle distribution present on fracture face A of plasma membranes of 3T3 Balb (Figs. 6-8) and SV-Balb (Figs. 9-11) cells incubated in phosphate-buffered saline after EDTA-mediated cell suspension. X50,000.
be positively identified as gap junctions (Figs. 1-3). These junctions were present either in normal or transformed cells and were more common in SV-Balb than 3T3 Balb. However, the higher incidence of junctions in cultures of transformed cells, which parallels previous observations of secondary and Rous sarcoma virus-transformed chick embryo fibroblasts (P. Pinto da Silva and N. B. Gilula, unpublished observations), cannot be considered significant because it might be due to: the higher incidence of regions of cell-to-cell apposition in cultures of transformed cells; and to the different probability of fracture due to differences in overall plasma membrane topography and the situation of the gap junctions relative to the cell profile (e.g., junctions in apposed or not apposed cell regions). In most instances the gap junctions appeared as small clusters of particles (Figs. 1 and 3). Frequently, several gap junctions occurred in close proximity to each other, occasionally following linear array (Fig. 1). The diameter of gap junction particles is approximately 8 nm. Treatment of normal or transformed Balb/c or Swiss 3T3 cells in situ with Con A did not result in an altered pattern of particle distribution (Figs. 4 and 5).
Cells in Suspension. Random distribution of the plasma membrane particles was also observed in cells fixed im-
mediately after EDTA-mediated cell suspension. If, however, the cell suspensions were washed and incubated in phosphatebuffered saline at 370 for 30 min, aggregation of the plasma membrane particles was observed in a fraction not exceeding half of the cells (Figs. 6-11). In Balb cells aggregation was more frequent and, when the highest levels of aggregation were compared, more intense in SV-transformed cells (MSV not tested) than in normal cells (Figs. 8 and 11). However, in 3T3 Swiss and SV-Swiss fibroblasts, particle aggregation was only observed in a small fraction of the cells (about 10%), and no differences were observed in the incidence and intensity of particle aggregation between normal and transformed cells. In plasma membranes displaying clear particle aggregation, the entire particle population coaggregated into a distinct network, leaving in between regions devoid of particles or "subparticles" (Figs. 8 and 11). Exposure of Balb unfixed cells to glycerol-phosphate-buffered saline also resulted in a pattern of aggregation similar, although less clear, to that observed by incubation in phosphate-buffered saline. In no case did it result in an island-like clustered pattern of particle distribution similar to that previously reported (17). Treatment of 3T3 Balb, SV-Balb, 3T3 Swiss, and SVSwiss cell suspensions with Con A (10 and 100 ug/ml) in phosphate-buffered saline for 30 min at 37° did not result in
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altered patterns of particle distribution relative to the controls (i.e., cells incubated in phosphate-buffered saline for the same period at the same temperature).
DISCUSSION Our experiments show no apparent differences in the- pattern of distribution of membrane particles as revealed by freezefracture of the plasma membranes of normal and SV- or MSV-transformed 3T3 fibroblasts. The membrane particles are randomly distributed either on the plasma membranes of cells prefixed in situ or of cells fixed immediately after EDTAmediated cell suspension. The few particle clusters that are seen can be positively identified as gap junctions (20, 21). No differences are found in gap junction morphology between normal and transformed cells. Furthermore, treatment in situ of normal and transformed cells with Con A does not result in alteration of the pattern of particle distribution. The present results are consistent with our previous observations of secondary and Rous sarcoma virus-transformed fibroblasts (16). However, they are in disagreement with those reported by Scott et al. (17), which describe distinct clusters of membrane particles on the fracture faces of the plasma membranes in normal Balb/c 3T3 (clone A31) fibroblasts associated with the progressive establishment of cell contact. We have used cells from the same clone under subconfluent and confluent conditions and, because of the long time reported necessary for full confluency of the cell cultures (17), we also observed 3T3 Balb cultures kept for up to 15 days (i.e., 10 days after reaching confluency). In all cases we observed a random pattern of particle distribution. It is not clear whether in the above-mentioned study (17) cells in situ or in suspension were used. Furthermore, in this study cells were freeze-fractured, either unfixed or lightly fixed with glutaraldehyde (1% for 5 min at 370; ref. 22). It is, thus, possible that particle clustering may have resulted from the combined effect of prolonged cell exposure of unfixed (or insufficiently fixed) cells to phosphate-buffered saline and glycerol (13, 19) (impregnation in 25% glycerol is necessary to avoid damage during rapid freezing of the samples). However, in our experiments exposure for 30 min of unfixed cells in situ to phosphate-buffered saline or, of cell suspensions to phosphatebuffered saline and glycerol, did not result in an island-like clustered pattern of distribution of the membrane particles that was reported (17). Our experiments show that treatment with Con A of normal or transformed 3T3 cells in situ does not result in alteration in the pattern of distribution of the membrane particles. However, in cells of the same clone and under very similar experimental conditions, exposure to Con A causes pronounced clustering of the Con A receptors in transformed (5, 6) and possibly also normal 3T3 cells (7). Taken together, these results imply that no direct structural relationship exists between membrane particles and Con A receptors. It is clear, however, that our experiments per se cannot independently establish this point. Independent distribution of membrane particles and Con A receptors is consistent with our recent observation of Entamoeba histolytica trophozoites (13, 14) and contrasts with our observation of a direct association of membrane intercalated particles and Con A receptors in erythro-
Proc. Nat. Acad. Sci. USA 72 (1975) cyte ghost membranes (12). At present, we cannot explain the aggregation of membrane particles that we observed in a fraction of the cells in suspension upon further incubation, but a recent report makes it likely that they represent stages in cell damage (23). In conclusion, our results show basic similarity of the freeze-fracture morphology of the plasma membranes of normal and transformed 3T3 cells, fail to demonstrate a specific effect of Con A molecules on the pattern of distribution of plasma membrane particles of normal and transformed cells, and, taken with previous evidence (5-7), imply independence of membrane particles and Con A receptors in transformed cells. We thank Dr. Dieter Paul for discussions and cell cultures, Miss Monique Lacorbiere for cell cultures, and Miss Adele Brodginski for preparation of the manuscript.. Work supported in part by Special Grant no. 638 from the California Division of the American Cancer Society, USPHS Research Grant no. CA15114 from the National Cancer Institute and a Core Grant to The Salk Institute (CA-14195) (to P.P.S.) and the Hoetchst Laboratories (to A.M-P.). 1. 2. 3. 4. 5.
6. 7. 8.
Burger, M. M. (1971) Curr. Top. Cell. Regul. 3,,135-193. Nicolson, G. L. (1971) Nature New Biol. 233, 244-246. Nicolson, G. L. (1972) Nature New Biol. 239, 193-197. Martinez-Palomo, A., Wicker, R. & Bernhard, W. (1972) Int. J. Cancer 9, 676-684. Rosenblith, J. Z., Ukena, T. E., Yin, H. H., Berlin, R. D. & Karnovsky, M. J. (1973) Proc. Nat. Acad. Sci. USA 70, 1625-1629. Nicolson, G. L. (1973) Nature New Biol.. 243, 218-220. de Petris, S., Raff, Ml. C. & Mallucci, L. (1973) Nature New Biol. 244, 275-278. Pinto da Silva, P. & Branton, D. (1970) J. Cell Biol. 45,
598-605.
9. Pinto da Silva, P., Branton, 1). & l)ouglas, S. 1). (1971) Nature 232, 194-196. 10. Tillack, T. W., Scott, R. E. & Marchesi, V. T. (1972) J. Exp. Med. 135, 1209-1227. 11. Pinto da Silva, P., Moss, P. S. & Fudenberg, H. H. (1973) Exp. Cell Res. 81, 127-138. 12. Pinto da Silva, P. & Nicolson, G. L. (1974) Biochim. Biophys. Acta 363, 311-319. 13. Pinto da Silva, P. & Martinez-Palomo, A. (1974) Nature 249, 170-171. 14. MNartinez-Palomo, A. & Pinto da Silva, P. (1974) in Perspectives in Membrane Biology, eds. Gitler, C. & Estrada, S. (Academic Press, New York), in press. 15. Karnovsky, M. J. & Unanue, E. R. (1973) Fed. Proc. 32, 55-59. 16. Pinto da Silva, P. & Gilula, N. B. (1972) Exp. Cell Res. 71,
393-401.
17. Scott, R. E., Furcht, L. T. & Kersey, J. H. (1973) Proc. Narat. Acad. Sci. USA 70, 3631-3635. 18. Guefin, C., Zachowski, A., Prigent, B., Paraf, A., Dunia, I., Diawara, WI. & Benedetti, E. L. (1974) Proc. Nat. Acad. Sci. USA 71, 114-117. 19. McIntyre, J. A., Gilula, N. B. & Karnovsky, M. J. (1974) J. Cell Biol. 60, 192-203. 20. Goodenough, D. A. & Revel, J. P. (1970) J. Cell Biol. 45, 272-290. 21. MIcNutt, N. S. & Weinstein, R. S. (1970) J. Cell Biol. 47, 666-688. 22. Barnett, R. E., Furcht, L. T. & Scott, R. E. (1974) Proc. Nat. Acad. Sci. USA 71, 1992-1994. 23. Cunningham, W. P., Staehelin, A. L., Rubin, R. W., Wilkins, R. & Bonneville, AM. (1974) J. Cell Biol. 62, 491504.
Distribution of Membrane Particles and Gap Junctions in Normal and Transformed 3T3 Cells Studied In Situ, in Suspension, and Treated with Concanavalin A (freeze-fracture/cancer/electron microscopy/viral transformation/cell culture)
PEDRO PINTO DA SILVA* AND ADOLFO MARTINEZ-PALOMOt * The Salk Institute for Biological Studies, San Diego, California 92112; and t Centro de Investigacion del I.P.N., Mexico 14, D.F., Mexico
Communicated by Robert W. Holley, November 29, 1974 Freeze-fracture techniques were used to AB STRACT study the ultrastructural distribution of plasma membrane particles in cultures of normal Balb/c and Swiss 3T3, and simian virus 40- or murine sarcoma virus-transformed fibroblasts. No apparent differences were observed. Cultures fixed in situ show a seemingly random distribution of membrane particles both in normal or in transformed cells. Treatment of cell cultures in situ with concanavalin A does not result in an altered pattern of particle distribution. EDTA-induced suspension of normal or transformed cells does not result, per se, in modification of the type of membrane particle distribution seen in cells fixed in situ. However, upon further incubation, a proportion of normal or transformed cells in suspension show a varying degree of particle aggregation following a network pattern. Concanavalin A treatment of normal and transformed cells in suspension does not result in a specific change of the pattern of particle distribution. Because it has been established that treatment with concanavalin A of simian virus 40-transformed Balb/c 3T3 fibroblasts causes pronounced clustering of the concanavalin A receptors at the outer surface, our results probably imply independence of membrane particles and concanavalin A receptors of these transformed cells.
receptors may also occur in normal Balb/c 3T3 fibroblasts
(7).
Unlike conventional thin-section electron microscope studies, freeze-fracture techniques allow further observation of the inner structure of biological membranes. During fracture the frozen membrane is split along the central plane of the bilayer, creating faces that contain numerous particles (8). Studies of the chemistry and topology of the particles have been mostly restricted to isolated erythrocyte membranes. In these membranes, combination of freeze-fracture and freeze-etch (sublimation) techniques has shown that the particles represent protein-containing structures that are intercalated and traverse the bilayer domain and expose antigens, lectin, and viral receptors and acidic sites at the outer surface (9-12). It is unlikely, however, that conclusions derived from the study of isolated erythrocyte membranes can be freely extrapolated as a model for the plasma membranes of live, eukaryotic cells. We have recently shown that on the plasma membranes of Entamoeba histolytica trophozoites, surface receptors may not be associated with the particles, since particles and surface receptors may move independently (13, 14). Independence of particles and Con A receptors has also been suggested in lymphocyte plasma membranes (15). This means that fluidity of the membrane can be considered at two separate levels: peripheral membrane fluidity (displayed by peripheral membrane components) and integral membrane fluidity (displayed by integral membrane components, such as those represented by the membrane particles). In the erythrocyte membrane, these concepts intersect because the membrane particles represent integral membrane components that are expressed as antigens and receptors at the outer membrane surface and, in consequence, movement of the particles implies movement of surface receptors
In recent years much attention has been devoted to the study of the composition, structure, and fluidity of the plasma membranes of normal and transformed cells. The use of these cell systems as experimental models of normal and cancerous growth stems, to a large extent, from the fact that while cultures of normal cells exhibit density-dependent inhibition of growth, in cultures of transformed cells proliferation continues after confluency (1). In initial studies of the surface distribution of concanavalin A (Con A) receptors in normal and transformed cells, it was concluded that the inherent distribution of these receptors was random. in normal cells while it was clustered or discontinuous in transformed cells (2-4). In these studies the membranes were either insufficiently fixed or not fixed before treatment with the lectin, and it was later shown that while the native distribution of Con A sites is random in both normal and transformed Balb/c 3T3 fibroblasts, treatment with Con A induces clustering of the receptors on the transformed cells (5, 6). Recently it has been reported that clustering of Con A
(9-12).
Freeze-fracture observations of the plasma membranes of secondary explant fibroblasts and of fibroblasts transformed by Rous sarcoma virus revealed random distribution in normal and transformed cells, as no clustering of particles was observed except for that which corresponded to gap junctions between normal or transformed cells (16). Recently, however, it was reported that in normal 3T3 fibroblasts (but not in cells transformed by simian virus 40) an intensely clustered distribution of the plasma membrane particles occurred upon the establishment of cell contacts, the incidence of which peaked as the cultures became confluent (17). Clustering of membrane particles was also reported in murine plasmacytoma cells treated with Con A and thought to represent,
Abbreviations: Con A, concanavalin A; 3T3 Balb, Balb/c 3T3 clone A31 fibroblasts; 3T3 Swiss, Swiss 3T3 clone A4 fibroblasts; SV- or MSV-Balb, Balb/c 3T3 clone A31 fibroblasts transformed by simian virus 40 or murine sarcoma virus, respectively; SVSwiss, Swiss 3T3 clone A4 fibroblasts transformed by simian virus 40.
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FIGS. 1-3. Fracture faces of the plasma membranes of 3T3 Balb (Fig. 1) and SV-Balb (Figs. 2 and 3) cells fixed in situ. With the exception of gap junctions (unusually numerous in Fig. 1), the particles are randomly distributed. Two loose particle clusters (Fig. 1, arrows) may represent stages of junction formation or disintegration. Gap junctions may appear in groups (Fig. 1) or isolated (Fig. 3, arrow). Figs. 1 and 3, X55,000; Fig. 2, X150,000.
in these cells, an acquired feature of the plasma membrane accompanying Con A-induced agglutination (18). In this study we have used Balb/c and Swiss 3T3 fibroblasts, normal and transformed by simian virus 40 (SV 40)
and murine sarcoma virus (\ISV), and report the absence of differences in the native pattern of distribution of the membrane particles as well as the absence of alteration in the pattern of distribution of membrane particles of normal and
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FIGs. 4 and 5. Plasma membrane fracture face A of 3T3 Balb (Fig. 4) and SV-Balb (Fig. 5) cells treated in situ with Con A. The pattern of particle distribution is not altered. X60,000. transformed cells treated with Con A. Gap junctions identified in cultures of normal or of transformed cells.
are
MATERIALS AND METHODS
Cells. Untransformed and SV40- or MSV-transformed Balb/c 3T3 A31, and untransformed Swiss 3T3 A4 and SV40transformed cells were used. The cells were plated at densities of 1 to 3 X 103 cells per cm2 in 75-cm2 Falcon flasks and were confluent within 3-5 days. Saturation densities of untransformed cells were 3 to 5 X 104 cells per cm2. The cells were grown in Dulbecco's modified Eigle's medium supplemented with 10% (v/v) calf serum (heat-inactivated for Balb/c cultures) changed at 3-day intervals. Treatment. (a) Cells in situ: Cell cultures were fixed in situ by addition of 50% glutaraldehyde to the medium to a final concentration of 1.5% and incubated for 30 min at 370. In experiments designed to study the effect of Con A, cell cultures were washed three times in phosphate-buffered saline (pH 7.3) and then incubated with 10 ml of Con A (10 and 100 ,ug/ml) in phosphate-buffered saline for 30 min at 37°. Controls were incubated in phosphate-buffered saline without Con A for the same period. The cells were then fixed as above. (b) Cells in suspension: Cell suspensions were obtained by incubation in 2 mM EDTA in Ca2+, Mg2+-free phosphatebuffered saline for 30 min at 370 (18). The cells were either fixed immediately in 1.5% glutaraldehyde for 30 min at 37° or washed twice and incubated for 30 min at 370 in the presence or absence. of Con A (10 or 100 ,ug/ml). The cells were then fixed as at ove before glycerol impregnation. (c) Glycerol impregnation: Because glycerol impregnation of unfixed cells can cause alterations of the pattern of distribution of plasma membrane particles (13, 19), effective glutaraldehyde fixation is necessary before glycerol impregnation. After glutaraldehyde fixation, cells fixed were removed from the dish with a Teflon spatula. All cells were suspended in about 1 ml of phosphate-buffered saline, gradually impregnated by dropwise addition of 25% glycerol in phosphatebuffered saline at 370 over a period of 10 min, incubated for 30 min at 370, and cooled for 1 hr at room temperature. To test the effect of glycerol on unfixed cells in suspension, some
cell cultures were also incubated in glycerol-phosphate-buffered saline without glutaraldehyde prefixation. Freeze-Fracture. The cells were frozen in the liquid phase of partially solidified Freon 22 and freeze-fractured in Balzer's 501 device equipped with an electron gun for platinum-carbon evaporation. The replicas were recovered, cleaned, and observed with an Hitachi HU-12 electron microscope at 75 kV. The micrographs are mounted with shadow direction from the bottom; shadows are white. Only fracture faces of the plasma membranes are shown. The membranes are split during fracture, and A face represents the outer aspect of the inner half of the bilayered regions of the membrane plus associated particles that protrude into the outer half of the membrane (8); vice versa for B face. The word "random" is used here not in the mathematical sense but, arbitrarily, to describe that the distribution of membrane particles does not follow a readily recognizable pattern. RESULTS
Cells In Situ. Observation of the fracture faces of plasma membranes of 3T3 Balb, 3T3 Swiss, SV-Balb, SV-Swiss, and MSV-Balb cells from cultures fined with 1.5% glutaraldehyde for 30 min at 370 directly in the culture dish invariably revealed a random distribution of membrane particles (Figs.1 and 3). As seen in freeze-fractured plasma membranes of other eukaryotic cells, there is remarkable heterogeneity in the size of the membrane particles, as all transitions can be observed from a definite particle to a clear smooth region. Smaller rugosities ("subparticles," ref. 13) were best visualized over membrane regions that were exposed to a lower angle of shadow. Because a previous report proposed that clustering of the particles of 3T3 Balb progressively increased with the establishment of cell contacts (17), we observed these cells in subconfluent (about 70% occupation of substrate) and confluent states. Also, because of the long periods of culture required to reach cell confluency and maximal incidence of particle clustering (Fig. 2 of ref. 17), we also observed 15-dayold cell cultures, i.e., cells that remained in culture for 10 days after reaching confluency. In all cases the distribution of particles was random. The only particle clusters found could
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FIGS. 6-11. Patterns of particle distribution present on fracture face A of plasma membranes of 3T3 Balb (Figs. 6-8) and SV-Balb (Figs. 9-11) cells incubated in phosphate-buffered saline after EDTA-mediated cell suspension. X50,000.
be positively identified as gap junctions (Figs. 1-3). These junctions were present either in normal or transformed cells and were more common in SV-Balb than 3T3 Balb. However, the higher incidence of junctions in cultures of transformed cells, which parallels previous observations of secondary and Rous sarcoma virus-transformed chick embryo fibroblasts (P. Pinto da Silva and N. B. Gilula, unpublished observations), cannot be considered significant because it might be due to: the higher incidence of regions of cell-to-cell apposition in cultures of transformed cells; and to the different probability of fracture due to differences in overall plasma membrane topography and the situation of the gap junctions relative to the cell profile (e.g., junctions in apposed or not apposed cell regions). In most instances the gap junctions appeared as small clusters of particles (Figs. 1 and 3). Frequently, several gap junctions occurred in close proximity to each other, occasionally following linear array (Fig. 1). The diameter of gap junction particles is approximately 8 nm. Treatment of normal or transformed Balb/c or Swiss 3T3 cells in situ with Con A did not result in an altered pattern of particle distribution (Figs. 4 and 5).
Cells in Suspension. Random distribution of the plasma membrane particles was also observed in cells fixed im-
mediately after EDTA-mediated cell suspension. If, however, the cell suspensions were washed and incubated in phosphatebuffered saline at 370 for 30 min, aggregation of the plasma membrane particles was observed in a fraction not exceeding half of the cells (Figs. 6-11). In Balb cells aggregation was more frequent and, when the highest levels of aggregation were compared, more intense in SV-transformed cells (MSV not tested) than in normal cells (Figs. 8 and 11). However, in 3T3 Swiss and SV-Swiss fibroblasts, particle aggregation was only observed in a small fraction of the cells (about 10%), and no differences were observed in the incidence and intensity of particle aggregation between normal and transformed cells. In plasma membranes displaying clear particle aggregation, the entire particle population coaggregated into a distinct network, leaving in between regions devoid of particles or "subparticles" (Figs. 8 and 11). Exposure of Balb unfixed cells to glycerol-phosphate-buffered saline also resulted in a pattern of aggregation similar, although less clear, to that observed by incubation in phosphate-buffered saline. In no case did it result in an island-like clustered pattern of particle distribution similar to that previously reported (17). Treatment of 3T3 Balb, SV-Balb, 3T3 Swiss, and SVSwiss cell suspensions with Con A (10 and 100 ug/ml) in phosphate-buffered saline for 30 min at 37° did not result in
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altered patterns of particle distribution relative to the controls (i.e., cells incubated in phosphate-buffered saline for the same period at the same temperature).
DISCUSSION Our experiments show no apparent differences in the- pattern of distribution of membrane particles as revealed by freezefracture of the plasma membranes of normal and SV- or MSV-transformed 3T3 fibroblasts. The membrane particles are randomly distributed either on the plasma membranes of cells prefixed in situ or of cells fixed immediately after EDTAmediated cell suspension. The few particle clusters that are seen can be positively identified as gap junctions (20, 21). No differences are found in gap junction morphology between normal and transformed cells. Furthermore, treatment in situ of normal and transformed cells with Con A does not result in alteration of the pattern of particle distribution. The present results are consistent with our previous observations of secondary and Rous sarcoma virus-transformed fibroblasts (16). However, they are in disagreement with those reported by Scott et al. (17), which describe distinct clusters of membrane particles on the fracture faces of the plasma membranes in normal Balb/c 3T3 (clone A31) fibroblasts associated with the progressive establishment of cell contact. We have used cells from the same clone under subconfluent and confluent conditions and, because of the long time reported necessary for full confluency of the cell cultures (17), we also observed 3T3 Balb cultures kept for up to 15 days (i.e., 10 days after reaching confluency). In all cases we observed a random pattern of particle distribution. It is not clear whether in the above-mentioned study (17) cells in situ or in suspension were used. Furthermore, in this study cells were freeze-fractured, either unfixed or lightly fixed with glutaraldehyde (1% for 5 min at 370; ref. 22). It is, thus, possible that particle clustering may have resulted from the combined effect of prolonged cell exposure of unfixed (or insufficiently fixed) cells to phosphate-buffered saline and glycerol (13, 19) (impregnation in 25% glycerol is necessary to avoid damage during rapid freezing of the samples). However, in our experiments exposure for 30 min of unfixed cells in situ to phosphate-buffered saline or, of cell suspensions to phosphatebuffered saline and glycerol, did not result in an island-like clustered pattern of distribution of the membrane particles that was reported (17). Our experiments show that treatment with Con A of normal or transformed 3T3 cells in situ does not result in alteration in the pattern of distribution of the membrane particles. However, in cells of the same clone and under very similar experimental conditions, exposure to Con A causes pronounced clustering of the Con A receptors in transformed (5, 6) and possibly also normal 3T3 cells (7). Taken together, these results imply that no direct structural relationship exists between membrane particles and Con A receptors. It is clear, however, that our experiments per se cannot independently establish this point. Independent distribution of membrane particles and Con A receptors is consistent with our recent observation of Entamoeba histolytica trophozoites (13, 14) and contrasts with our observation of a direct association of membrane intercalated particles and Con A receptors in erythro-
Proc. Nat. Acad. Sci. USA 72 (1975) cyte ghost membranes (12). At present, we cannot explain the aggregation of membrane particles that we observed in a fraction of the cells in suspension upon further incubation, but a recent report makes it likely that they represent stages in cell damage (23). In conclusion, our results show basic similarity of the freeze-fracture morphology of the plasma membranes of normal and transformed 3T3 cells, fail to demonstrate a specific effect of Con A molecules on the pattern of distribution of plasma membrane particles of normal and transformed cells, and, taken with previous evidence (5-7), imply independence of membrane particles and Con A receptors in transformed cells. We thank Dr. Dieter Paul for discussions and cell cultures, Miss Monique Lacorbiere for cell cultures, and Miss Adele Brodginski for preparation of the manuscript.. Work supported in part by Special Grant no. 638 from the California Division of the American Cancer Society, USPHS Research Grant no. CA15114 from the National Cancer Institute and a Core Grant to The Salk Institute (CA-14195) (to P.P.S.) and the Hoetchst Laboratories (to A.M-P.). 1. 2. 3. 4. 5.
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