Cell Biology
International
Reports,
Vol. 7, No. 5, 1977
469
FIBRONECTIN MATRIX: ANTIBODY-INDUCED REORGANIZATION IN HUMAN FIBROBLAST CULTURES Markku Kurkinen' and Antti Vaheri Department of Virology, University of Helsinki Haartmaninkatu 3, 00290 Helsinki 29, Finland ' To whom correspondence
should
be addressed
SUMMARY Fibronectin, a major pericellular glycoprotein of adherent cells, was predominantly present in fibrillar structures in human fibroblast cultures as shown by indirect immunofluorescence. In conventional "patching experiments" where one day old cells were expos:d to anti-fibronectin IgG in the cold, washed, and reincubated at 37 no redistribution was seen. However, continuous exposure of the cultures to IgG at 37o resulted in redistribution. The fibrillar structures were lost and fibronectin aggregates (patches) were founu. Fab-fragments had no such effect. These results support the findings that fibronectrn is predominantly a matrix protein and show that matrix components may be redistributed in cell culture conditions. INTRODUCTION Fibronectin (Vaheri et al., 19761, also known as LETS protein (Hynes, 1973) or CSP (Yamada and Weston, 1974) is a major pericellular glycoprotein (subunit mol.wt. 220 000). Both normal and transformed Eibroblasts synthesize and secrete the protein but the latter have lost the ability to form a pericellular fibronectin matrix (Vaheri and Ruoslahti, 1975; Hedman et ai., in press). We report here that divalent anti-fibronectin immunoglobulin induces redistribution of pericellular fibronectin in cultures of normai adherent human fibroblasts. MATERIAL AND METHODS either locally established or comHuman fibroblast strains, mercially obtained (WI-38 cells from American Type Culture Collection, Rockville, Md.) were grown in plastic tissue culture dishes at 37' in Eagle's basal medium (diploid) supplemented with 10 z foetal calf serum, penicillin, 100 U/ml, and streptomycin, 50 ug/ml. Stock cultures of each strain were tested biweekly for For the mycoplasma (Russel et al., 1975) with negative results. experiments cells from confluent cultures were seeded in a I:2 ratio to dishes with glass coverslips. Goat and rabbit antisera against human fibronectin purified from plasma were prepared as described by Keski-Oja et al. (1976). The antisera were absorbed with lyophilized foetal calf serum and did not react with the bovine fibronectin in the culture medium; The antibodies were monospecific as judged by immunodiffusion against normal human plasma, double antibody immunoprecipitation tests against human cell extracts. by
470
Cell Biology
International
Reports,
Vol. 1, No. 5, 19 77
blocking experiments and by SDS-polyacrylamide electrophoresis of the immunizing antigen, as documented elsewhere (Stenman et al., in press). Fab-fragments were prepared according to Porter (1959). anti-goat IgG and isothiocyanate (FITC)-conjugated Fluorescent anti-rabbit IgG were from Nordic Immunology, London, and Wellcome, A Zeiss fluorescence microscope Beckenham, England, respectively. with epi-illuminator III RS equipped with filters and beam splitter for specific FITC-fluorescence and oil immersion objective with high numerical aperture (1.4) were used. RESULTS When cultures of normal human embryonic lung fibroblasts were fixed and stained for external fibronectin by indirect immunofluorescence a characteristic fibrillar distribution was seen (Fig. la Similar organization was observed if live cells were and d). in agreement with earlier findings stained in the cold (Fig. le), exposure of live cells to (Wartiovaara et al., 1974). In contrast, anti-fibronectin antibodies for one hour at 37'C with subsequent fixation and staining with the FITC-conjugate resulted in a The strikingly different organization of external fibronectin. visually estimated amount of fibrillar fibronectin was reduced by about 75 % (Fig. lb) and numerous aggregates or patches of variable size were present (Fig. lc). No gross change in cell morphology was seen in phase or in Nomarski microscopy. The patches were found predominantly on the under surface of the cell at the substrate level and were distributed throughout the cell. The results (presented here for HE6L embryonic lung fibroblasts and anti-fibronectin goat antibodies) were identical when human embryonic skin (HES-LA), adult skin (ES, JKO) or embryonic lung (WI-38) fibroblasts and when rabbit antibodies were used. Furthermore, the pattern was the same whether the antibody treated cells were re-exposed to the antibody after fixation or stained directly with the conjugate. Neither cycloheximide (10 ug/ml) nor colcemide (100 rig/ml) had any effect on the antibody induced patching. In these experiments the drug treatments were initiated 30 min prior to exposure of the cells to the antibody. Under these conditions cyclohex$mide inhibited protein synthesis by about 90 % as judged by H-leucine incorporation into acid-insoluble form. The above dose of colcemide resulted in some rounding of the cells during the experiments, and prolonged exposure in serum containing medium resulted in arrest of the fibroblasts at metaphase. The antibody-induced patches were observed only when live cells were exposured to divalent antibodies at 37'C. Exposure to Fabfragments at 37'C had not effect (Fig. If). If live cells were first treated with the antibody for 20 or 60 min at 4'C, washed free of unbound antibody and then incubated in fresh medium at 37'C for further 60 minutes, fibrils but not patches were seen after immunofluorescent staining. The arstibody-induced patching could be readily observed in young, 1-2 day old cultures. If confluent fibroblast cultures (4-6 days old) were used, antibody-induced patching of external fibronectin still occurred but was difficult to assay because of the
Cell Biology
International
Reports,
Vol. 1, No. 5, 19 7 7
471
Fi gure 1. Antibody-induced n redistribution of external fibronecti de tected by indirect immunofluorescence. a, d: cultures fixed wi th 3. 5 % formaldehyde (in phosphate buffered saline, PBS), and incub ated wi th antibodies for 60 minutes at 37'C. b, c: live cells incubat ed wi th antibodies for 60 minutes at 4'C. f: live cells incubated wi th antibody Fab-fragments for 60 minutes at 37'C. g: one week old cu ltures fixed with formaldehyde, reacted with antibodies for 60 mi nutes at 37'C and stained. Scales denote 50 urn (a, b) and 10 urn cc -g) * Two day old human embryonic (HEGL strain) lung fibroblasts own on coverslips were rinced three times with warm Eagle's bas al gr
472
Cell Biology
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Reports,
Vol. 1, No. 5, 79 7 7
Figure 1. (continued) medium (diploid) containing 0.05 % bovine serum albumin, 100 units/ml penicillin, 50 ug/ml of streptomycin and 10 mM of Hepes (N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid). After equilibration (15 minutes) at the temperature indicated anti-fibronectin goat IgG or Fab-fragments were added to a final protein concentrations of 0.1 mg/ml and 0.3 mg/ml, respectiveThe cultures were then incubated at the temperature indicated ly. for 60 minutes, rinsed three times with Dulbecco's phosphate buffered saline at the same temperature, and fixed with 3.5 % formaldehyde in PBS for 20 minutes at room temperature. The coverslips were then stained for 30 minutes with anti-goat FITC-IgG at room temperature, washed with PBS, mounted wet in 50 % glycerol in isotonic Verona1 buffer saline, pH 8.6, and examined by fluorescent microscopy.
Cell Biology
International
Reports,
Vol. 1, No. 5, 7977
473
dense extracellular fibrillar matrix of fibronectin which characteristically develops around cells in confluent cultures in vitro (Fig. lg) (Wartiovaara et al., 1974). DISCUSSION These experiments show for the first time that exposure of cultures of normal adherent fibroblasts to divalent antibodies results in the reorganization and patchy distribution of a surface In interpreting this finding it is crusial to recognize protein. that fibronectin is not a conventional membrane protein. In cell surface-iodinated fibronectin from hamster fractination experiments, fibroblasts does not copurify with plasma membranes (Marciagi and from a dense (y = 1.25 g/cm ) Bader, 1975), but is recovered Immunoelectron microparticulate fraction (Graham et al., 1975). scopy of adherent human fibroblasts with either peroxidase or ferritin conjugate has shown that even in young (1 day old) cultures most external fibronectin is present as a pericellular Only a small fraction is found fibrillar "in vitro matrix". membrane-associated (Hedman et al., in press). In conventional "patching experiments" that have been used to demonstrate ligand-induced lateral mobility of membrane components, This suggests that no redistribution of fibronectin was detected. the membrane-associated fibronectin may have a restricted lateral mobility on the membrane. Most of the previous studies on redistribution of cell surface components (Taylor et al., 1971; Edidin, 1974; Raff and de Petris, 1973; Nicolson, 1976) have been carried out using suspended cells, is relevant only to hematowhich condition, strictly speaking, From studies on adherent fibroblastic cells, poietic cell types. it has been concluded that neither H-2 antigens (Edidin and Weiss, 1974) nor Concanavalin A receptors (Ukena et al., 1974) could be redistributed unless the cells were protease-treated or virustransformed. we see the re-organization of pericellular In conclusion, fibronectin as induced by excess antibody at 37'C indicating that matrix components in cell culture conditions are not fixed but can be redistributed by ligands. This We thank Mrs. Riitta RBty for technical assistance. research was supported by grants from the US National Institutes of Health (CA-17373) and the Finnish Cultural Foundation. REFERENCES and translational diffusion in memM. (1974). Rotational branes. Annual Review of Biophysics and Bioengineering 3, 179-202. Restriction of antigen mobility Edidin, M. and Weiss, R. (1974). in the plasma membranes of some cultured fibroblasts. In: Control of Proliferation B. Clarkson and R. Baserga (eds.). in Animal Cells. pp. 213-219. Cold Spring Harbor Laboratory.
Edidin,
474
Cell Biology
International
Reports,
Vol. 1, No. 5, 1977
Graham, J.H., Hynes, R.O., Davidson, E.A. andll&inton, D.F. (1975). The location of proteins labeled by the I-lactoperoxidase system in the NIL 8 hamster fibroblast. Cell $, 353-365. Hedman, K., Vaheri, A. and Wartiovaara, J. (1977). Cell surface protein, fibronectin, of human fibroblast cultures, has a membrane-associated and a predominant pericellular matrix form. Journal of CellBiology, in press. Hynes, R.O. (1973). Alteration of cell-surface proteins by viral transformation and by proteolysis. Proceedings of National Academy of Sciences USA 70, 3170-3174. Cross-linking Keski-Oja, J., Mosher, D.F. aa Vaheri, A. (1976). of a major fibroblast surface-associated protein (fibronectin) catalyzed by blood coagulation factor XIII. Cell 2, 29-35. Polypeptide composition of Marciani, D.J. and Bader, J.P. (1975). cell membranes from chick embryo fibroblasts transformed by Rous sarcoma virus. Biochimica Biophysics Acta 5, 386-398. Nicolson, G.L. (1976). Trans-membrane control over the receptors Surface changes associated with on normal and tumor cells. II. transformation and malignancy. Biochimica Biophysics Acta -'458 l-72. Porter, R.R. (1959). The hydrolysis of rabbit gamma globulin and antibodies with crystalline papain. Biochemical Journal -'73 119-126. Raff, M.C. and de Petris, S. (1973). Movement of lymphocyte surface antigens and receptors: the fluid nature of the lymphocyte plasma membrane and its immunological significance. Federation Proceedings 32, 48-54. Russel, W.C., Newman, C. and Williamson, D.H. (1975). A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature 253, 461-462. Stenman, S., Wartiovaara, J. and Vaheri, A. (1977)TChanges in the distribution of a major fibroblast protein,fibronectin, during mitosis and interphase. Journal of Cell Biology, in press. Taylor, R.B., Duffus, P.H., Raff, M.C. and de Petris, S. (1971). Redistribution and pinocytosis of lymphocyte surface immunoglobulin molecules induced by anti-immunoglobulin antibody. NatureNew Biology 233, 225-229. Ukena, T.E., Borysenko, J.Z., Karnovsky, M.J. and Berlin, R.D. (1974). Effects of colchicine, cytochalasin B, and Z-deoxyglucose on the topographical organization of surface-bound concanavalin A in normal and transformed fibroblasts. Journal of Cell Biology 61, 70-82. Wartiovaara, J., Linder, E., Ruoslahti, E. and Vaheri, A. (1974). Distribution of fibroblast surface antigen. Association with fibrillar structures of normal cells and loss upon viral transformation. Journal of Experimental Medicine 140, 1522-1533, Vaheri, A. and Ruoslahti, E. (1974). Disappearance of a major celltype specific surface antigen (SF) after transformation of fibroblasts by Rous sarcoma virus. International Journal of Cancer 2, 579-586. Vaheri, A., Ruoslahti, E., Linder, E., Wartiovaara, J., Keski-Oja, J ., Kuusela, P. and Saksela, 0. (1976). Molecular properties,
Cell Biology
International
Reports,
Vol. 1, No. 5, 1977
475
distribution in vitro and in vivo, and altered expression in transformed cells. Journal of Supramolecular Structure 4, 63-70. Isolation of a major cell Yamada, K.M. and Weston, J.A. (1974). Proceedings of National surface glycoprotein from fibroblasts. Academy of Sciences USA 71, 3492-3496.
International
Reports,
Vol. 7, No. 5, 1977
469
FIBRONECTIN MATRIX: ANTIBODY-INDUCED REORGANIZATION IN HUMAN FIBROBLAST CULTURES Markku Kurkinen' and Antti Vaheri Department of Virology, University of Helsinki Haartmaninkatu 3, 00290 Helsinki 29, Finland ' To whom correspondence
should
be addressed
SUMMARY Fibronectin, a major pericellular glycoprotein of adherent cells, was predominantly present in fibrillar structures in human fibroblast cultures as shown by indirect immunofluorescence. In conventional "patching experiments" where one day old cells were expos:d to anti-fibronectin IgG in the cold, washed, and reincubated at 37 no redistribution was seen. However, continuous exposure of the cultures to IgG at 37o resulted in redistribution. The fibrillar structures were lost and fibronectin aggregates (patches) were founu. Fab-fragments had no such effect. These results support the findings that fibronectrn is predominantly a matrix protein and show that matrix components may be redistributed in cell culture conditions. INTRODUCTION Fibronectin (Vaheri et al., 19761, also known as LETS protein (Hynes, 1973) or CSP (Yamada and Weston, 1974) is a major pericellular glycoprotein (subunit mol.wt. 220 000). Both normal and transformed Eibroblasts synthesize and secrete the protein but the latter have lost the ability to form a pericellular fibronectin matrix (Vaheri and Ruoslahti, 1975; Hedman et ai., in press). We report here that divalent anti-fibronectin immunoglobulin induces redistribution of pericellular fibronectin in cultures of normai adherent human fibroblasts. MATERIAL AND METHODS either locally established or comHuman fibroblast strains, mercially obtained (WI-38 cells from American Type Culture Collection, Rockville, Md.) were grown in plastic tissue culture dishes at 37' in Eagle's basal medium (diploid) supplemented with 10 z foetal calf serum, penicillin, 100 U/ml, and streptomycin, 50 ug/ml. Stock cultures of each strain were tested biweekly for For the mycoplasma (Russel et al., 1975) with negative results. experiments cells from confluent cultures were seeded in a I:2 ratio to dishes with glass coverslips. Goat and rabbit antisera against human fibronectin purified from plasma were prepared as described by Keski-Oja et al. (1976). The antisera were absorbed with lyophilized foetal calf serum and did not react with the bovine fibronectin in the culture medium; The antibodies were monospecific as judged by immunodiffusion against normal human plasma, double antibody immunoprecipitation tests against human cell extracts. by
470
Cell Biology
International
Reports,
Vol. 1, No. 5, 19 77
blocking experiments and by SDS-polyacrylamide electrophoresis of the immunizing antigen, as documented elsewhere (Stenman et al., in press). Fab-fragments were prepared according to Porter (1959). anti-goat IgG and isothiocyanate (FITC)-conjugated Fluorescent anti-rabbit IgG were from Nordic Immunology, London, and Wellcome, A Zeiss fluorescence microscope Beckenham, England, respectively. with epi-illuminator III RS equipped with filters and beam splitter for specific FITC-fluorescence and oil immersion objective with high numerical aperture (1.4) were used. RESULTS When cultures of normal human embryonic lung fibroblasts were fixed and stained for external fibronectin by indirect immunofluorescence a characteristic fibrillar distribution was seen (Fig. la Similar organization was observed if live cells were and d). in agreement with earlier findings stained in the cold (Fig. le), exposure of live cells to (Wartiovaara et al., 1974). In contrast, anti-fibronectin antibodies for one hour at 37'C with subsequent fixation and staining with the FITC-conjugate resulted in a The strikingly different organization of external fibronectin. visually estimated amount of fibrillar fibronectin was reduced by about 75 % (Fig. lb) and numerous aggregates or patches of variable size were present (Fig. lc). No gross change in cell morphology was seen in phase or in Nomarski microscopy. The patches were found predominantly on the under surface of the cell at the substrate level and were distributed throughout the cell. The results (presented here for HE6L embryonic lung fibroblasts and anti-fibronectin goat antibodies) were identical when human embryonic skin (HES-LA), adult skin (ES, JKO) or embryonic lung (WI-38) fibroblasts and when rabbit antibodies were used. Furthermore, the pattern was the same whether the antibody treated cells were re-exposed to the antibody after fixation or stained directly with the conjugate. Neither cycloheximide (10 ug/ml) nor colcemide (100 rig/ml) had any effect on the antibody induced patching. In these experiments the drug treatments were initiated 30 min prior to exposure of the cells to the antibody. Under these conditions cyclohex$mide inhibited protein synthesis by about 90 % as judged by H-leucine incorporation into acid-insoluble form. The above dose of colcemide resulted in some rounding of the cells during the experiments, and prolonged exposure in serum containing medium resulted in arrest of the fibroblasts at metaphase. The antibody-induced patches were observed only when live cells were exposured to divalent antibodies at 37'C. Exposure to Fabfragments at 37'C had not effect (Fig. If). If live cells were first treated with the antibody for 20 or 60 min at 4'C, washed free of unbound antibody and then incubated in fresh medium at 37'C for further 60 minutes, fibrils but not patches were seen after immunofluorescent staining. The arstibody-induced patching could be readily observed in young, 1-2 day old cultures. If confluent fibroblast cultures (4-6 days old) were used, antibody-induced patching of external fibronectin still occurred but was difficult to assay because of the
Cell Biology
International
Reports,
Vol. 1, No. 5, 19 7 7
471
Fi gure 1. Antibody-induced n redistribution of external fibronecti de tected by indirect immunofluorescence. a, d: cultures fixed wi th 3. 5 % formaldehyde (in phosphate buffered saline, PBS), and incub ated wi th antibodies for 60 minutes at 37'C. b, c: live cells incubat ed wi th antibodies for 60 minutes at 4'C. f: live cells incubated wi th antibody Fab-fragments for 60 minutes at 37'C. g: one week old cu ltures fixed with formaldehyde, reacted with antibodies for 60 mi nutes at 37'C and stained. Scales denote 50 urn (a, b) and 10 urn cc -g) * Two day old human embryonic (HEGL strain) lung fibroblasts own on coverslips were rinced three times with warm Eagle's bas al gr
472
Cell Biology
International
Reports,
Vol. 1, No. 5, 79 7 7
Figure 1. (continued) medium (diploid) containing 0.05 % bovine serum albumin, 100 units/ml penicillin, 50 ug/ml of streptomycin and 10 mM of Hepes (N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid). After equilibration (15 minutes) at the temperature indicated anti-fibronectin goat IgG or Fab-fragments were added to a final protein concentrations of 0.1 mg/ml and 0.3 mg/ml, respectiveThe cultures were then incubated at the temperature indicated ly. for 60 minutes, rinsed three times with Dulbecco's phosphate buffered saline at the same temperature, and fixed with 3.5 % formaldehyde in PBS for 20 minutes at room temperature. The coverslips were then stained for 30 minutes with anti-goat FITC-IgG at room temperature, washed with PBS, mounted wet in 50 % glycerol in isotonic Verona1 buffer saline, pH 8.6, and examined by fluorescent microscopy.
Cell Biology
International
Reports,
Vol. 1, No. 5, 7977
473
dense extracellular fibrillar matrix of fibronectin which characteristically develops around cells in confluent cultures in vitro (Fig. lg) (Wartiovaara et al., 1974). DISCUSSION These experiments show for the first time that exposure of cultures of normal adherent fibroblasts to divalent antibodies results in the reorganization and patchy distribution of a surface In interpreting this finding it is crusial to recognize protein. that fibronectin is not a conventional membrane protein. In cell surface-iodinated fibronectin from hamster fractination experiments, fibroblasts does not copurify with plasma membranes (Marciagi and from a dense (y = 1.25 g/cm ) Bader, 1975), but is recovered Immunoelectron microparticulate fraction (Graham et al., 1975). scopy of adherent human fibroblasts with either peroxidase or ferritin conjugate has shown that even in young (1 day old) cultures most external fibronectin is present as a pericellular Only a small fraction is found fibrillar "in vitro matrix". membrane-associated (Hedman et al., in press). In conventional "patching experiments" that have been used to demonstrate ligand-induced lateral mobility of membrane components, This suggests that no redistribution of fibronectin was detected. the membrane-associated fibronectin may have a restricted lateral mobility on the membrane. Most of the previous studies on redistribution of cell surface components (Taylor et al., 1971; Edidin, 1974; Raff and de Petris, 1973; Nicolson, 1976) have been carried out using suspended cells, is relevant only to hematowhich condition, strictly speaking, From studies on adherent fibroblastic cells, poietic cell types. it has been concluded that neither H-2 antigens (Edidin and Weiss, 1974) nor Concanavalin A receptors (Ukena et al., 1974) could be redistributed unless the cells were protease-treated or virustransformed. we see the re-organization of pericellular In conclusion, fibronectin as induced by excess antibody at 37'C indicating that matrix components in cell culture conditions are not fixed but can be redistributed by ligands. This We thank Mrs. Riitta RBty for technical assistance. research was supported by grants from the US National Institutes of Health (CA-17373) and the Finnish Cultural Foundation. REFERENCES and translational diffusion in memM. (1974). Rotational branes. Annual Review of Biophysics and Bioengineering 3, 179-202. Restriction of antigen mobility Edidin, M. and Weiss, R. (1974). in the plasma membranes of some cultured fibroblasts. In: Control of Proliferation B. Clarkson and R. Baserga (eds.). in Animal Cells. pp. 213-219. Cold Spring Harbor Laboratory.
Edidin,
474
Cell Biology
International
Reports,
Vol. 1, No. 5, 1977
Graham, J.H., Hynes, R.O., Davidson, E.A. andll&inton, D.F. (1975). The location of proteins labeled by the I-lactoperoxidase system in the NIL 8 hamster fibroblast. Cell $, 353-365. Hedman, K., Vaheri, A. and Wartiovaara, J. (1977). Cell surface protein, fibronectin, of human fibroblast cultures, has a membrane-associated and a predominant pericellular matrix form. Journal of CellBiology, in press. Hynes, R.O. (1973). Alteration of cell-surface proteins by viral transformation and by proteolysis. Proceedings of National Academy of Sciences USA 70, 3170-3174. Cross-linking Keski-Oja, J., Mosher, D.F. aa Vaheri, A. (1976). of a major fibroblast surface-associated protein (fibronectin) catalyzed by blood coagulation factor XIII. Cell 2, 29-35. Polypeptide composition of Marciani, D.J. and Bader, J.P. (1975). cell membranes from chick embryo fibroblasts transformed by Rous sarcoma virus. Biochimica Biophysics Acta 5, 386-398. Nicolson, G.L. (1976). Trans-membrane control over the receptors Surface changes associated with on normal and tumor cells. II. transformation and malignancy. Biochimica Biophysics Acta -'458 l-72. Porter, R.R. (1959). The hydrolysis of rabbit gamma globulin and antibodies with crystalline papain. Biochemical Journal -'73 119-126. Raff, M.C. and de Petris, S. (1973). Movement of lymphocyte surface antigens and receptors: the fluid nature of the lymphocyte plasma membrane and its immunological significance. Federation Proceedings 32, 48-54. Russel, W.C., Newman, C. and Williamson, D.H. (1975). A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature 253, 461-462. Stenman, S., Wartiovaara, J. and Vaheri, A. (1977)TChanges in the distribution of a major fibroblast protein,fibronectin, during mitosis and interphase. Journal of Cell Biology, in press. Taylor, R.B., Duffus, P.H., Raff, M.C. and de Petris, S. (1971). Redistribution and pinocytosis of lymphocyte surface immunoglobulin molecules induced by anti-immunoglobulin antibody. NatureNew Biology 233, 225-229. Ukena, T.E., Borysenko, J.Z., Karnovsky, M.J. and Berlin, R.D. (1974). Effects of colchicine, cytochalasin B, and Z-deoxyglucose on the topographical organization of surface-bound concanavalin A in normal and transformed fibroblasts. Journal of Cell Biology 61, 70-82. Wartiovaara, J., Linder, E., Ruoslahti, E. and Vaheri, A. (1974). Distribution of fibroblast surface antigen. Association with fibrillar structures of normal cells and loss upon viral transformation. Journal of Experimental Medicine 140, 1522-1533, Vaheri, A. and Ruoslahti, E. (1974). Disappearance of a major celltype specific surface antigen (SF) after transformation of fibroblasts by Rous sarcoma virus. International Journal of Cancer 2, 579-586. Vaheri, A., Ruoslahti, E., Linder, E., Wartiovaara, J., Keski-Oja, J ., Kuusela, P. and Saksela, 0. (1976). Molecular properties,
Cell Biology
International
Reports,
Vol. 1, No. 5, 1977
475
distribution in vitro and in vivo, and altered expression in transformed cells. Journal of Supramolecular Structure 4, 63-70. Isolation of a major cell Yamada, K.M. and Weston, J.A. (1974). Proceedings of National surface glycoprotein from fibroblasts. Academy of Sciences USA 71, 3492-3496.
