- Email: [email protected]
Isolation of focal contact membrane using saponin
582 Short notes
SHORT NOTE Isolation of Focal Contact Membrane Using Saponin A. A. NEYFAKH, Laboratory
of Molecular
JR and T. M. SVITKINA
Biology and Bio-organic Chemistry, University, Moscow 117234, USSR
Moscow
State
The fragments of lower cell surface remained attached to the substrate after incubation of mouse or chick fibroblasts in 0.2% saponin solution and subsequent removal of cells under the action of shearing force. These fragments corresponded exactly to the cellular focal contacts seen by interference reflection microscopy. Ultrastructurally they were membrane fragments with typical three-layered structure. No cytoskeletal components were found in saponin-isolated focal contact membranes either by immunofluorescence or electron microscopy. Only one major cell-derived protein with an apparent molecular weight (MW) of 51 kD (chick embryo fibroblasts) or 47 kD (mouse embryo fibroblasts) remained on the substrate after saponin treatment and removal of cells.
In recent years considerable attention has been focused on the mechanism of cell attachment to the substrate, especially on the structure of focal contacts. Focal contacts are discrete regions of the lower cell surface, located most closely to the substrate, associated with the ends of microfilament bundles [l, 21 and enriched in vinculin [3, 41. In spite of numerous investigations the mechanisms of both membrane-to-substrate attachment and membrane-to-cytoskeleton association in focal contacts remain obscure. Chemical detachment or mechanical removal of cells were used for the purification of the components involved in cell adhesion [5, 61. Here we propose a modification of this approach which allows isolation of pure focal contact membranes . Materials
and Methods
Secondary cultures of mouse or chick embryo tibroblasts were cultivated on coverslips in the 1 : 1 mixture of medium 199 and 0.5 % lactalbumin hydrolysate, supplemented with 10% calf serum. For metabolic labelling chick embryo fibroblasts were grown in roller bottles until subconfluence and then cultivated overnight in serum-free Eagle’s medium, containing 25 @i/ml of 14Camino acids mixture (UVVVR). Proteins secreted by cells were precipitated from this medium with 10% trichloroacetate. For saponin treatment cells were washed with phosphate-buffered saline (PBS) and then incubated for 3-5 min in 0.2% saponin (Merck) in PBS or in cytoskeleton-stabilizing buffer M [7]. Then the cells grown on coverslips were intensively pipetted in the same solution. Roller bottles were vigorously shaken manually. For immunofluorescence staining, specimens were fixed with 4% formaldehyde in PBS. The staining procedure and antibodies to actin and vinculin are described elsewhere [7, 161.The monoclonal antibody to vimentin (clone S47D9) was a generous gift of Dr E B Mechetner. Fluorescence microscopy as well as interference reflection microscopy [8] were performed using Zeiss Photomicroscope III. For transmission electron microscopy (TEM) specimens were fixed with a mixture of 2 % glutaraldehyde and 0.4 % tannic acid in 0.1 M cacodylate buffer (pH 7.2), post-fixed in 1% OsO,, contrasted with uranyl acetate during the ethanol dehydration and embedded in Spurr’s resin [9]. Before ultrathin sectioning the coverslips were dissolved in hydrofluoric acid. Gel electrophoresis was performed according to Laemmli [lo] using 7.5-15 % gradient polyacrylamide slab gels. Labelled proteins were revealed by fluorography [ll]. Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/83 SO3.t?4
Fig. 1. Mouse embryo fibroblast (a, c) before; (b, d) after saponin treatment and pipetting. (a, b) Phase contrast; (c, d) interference reflection. Bar, 20 pm.
Results After incubation of substrate-spread mouse or chick fibroblasts in saponincontaining PBS or buffer M, cells were easily detached from the substrate by means of shearing force (pipetting or shaking). Only small hardly visible cell fragments remained on the glass surface (fig. 1 b). Using interference reflection microscopy [8] these fragments could be easily seen as black strips and spots (fig. 16). Their morphology and distribution were similar to those of focal contact regions in intact cells. The coincidence of cell fragments remaining on the glass and focal contacts was proved by photographing the cell before and after saponin treatment (compare fig. 1 c, 6). Practically all focal contacts remained on the glass, while other regions of the lower cell surface, looking grey or white on the interference reflection picture, were completely removed by sufficiently vigorous pipetting. The fragments remaining on glass, hereafter called ‘saponin-isolated focal contacts’, were examined for the presence of actin and vinculin. The focal contacts of intact cells were brightly stained with anti-vinculin and were found to be associated with the ends of actin bundles. After gentle pipetting, retaining some cells on the substrate, saponin-isolated contacts of removed cells could be Exp Cell Res I49 (19.33)
584 Short notes
Fig. 2. Ultrastructure of saponin-isolated focal contacts of mouse embryo tibroblasts. Note the triplelayered membrane, connected with the substrate by electron-dense bridges. Bottom, Membrane fragment with coated pits. TEM. Bar, 0.1 pm. Fig. 3. Fluorogram of a gel after electrophoresis of “C-labelled proteins. A, Lysate of chick embryo fibroblasts; B, proteins secreted by chick embryo fibroblasts into the medium; C, D, material remaining on the substrate after saponin treatment and removal of chick (C) or mouse (D) tibroblasts. The positions of r4C-labelled marker proteins (Amersham) are indicated.
stained with anti-vinculin. Occasionally the residues of actin bundles were also seen on the substrate. However, after intensive pipetting both actin and vinculin were absent in saponin-isolated focal contacts. As revealed by immunofluorescence staining the protein of intermediate filaments, vimentin, was also absent. The ultrastructure of saponin-isolated focal contacts was investigated by TEM of ultrathin sections perpendicular to the substrate (fig. 2). The substrate surface was identified by the continuous layer of glass-adsorbed serum proteins. Membrane fragments up to several micrometers in size with characteristic threelayered structure were observed closely (about IO-20 nm) to the substrate. Since no other cell-derived material was found on the sections these membranes were evidently saponin-isolated focal contacts. The membranes were connected with the serum protein layer by irregular bridging fibres. There were no structures on Exp Cell Res 149 (1983)
Short notes
585
the inner side of saponin-isolated membranes except occasional coated pits (fig. 2, bottom). In order to study the protein composition of saponin-isolated contacts chick fibroblasts were labelled with 14C-amino acids. The cultures were then treated with saponin and shaken. The material remaining on glass was dissolved in 0.2 % sodium dodecyl sulfate (SDS) and subjected to gel electrophoresis. Only one major protein band, corresponding to MW 51 kD, and a few faint bands were observed on the fluorogram of the gel (fig. 3, C). The addition of the protease inhibitor phenylmethylsulfonylfluoride to all solutions used did not alter the spectrum of labelled polypeptides. The 51 kD protein is not a major protein of intact cells (compare fig. 3, A, C). We have also shown that it is not secreted by fibroblasts into the medium (compare fig. 3, B, C). The secretion of three major polypeptides, which most likely represent fibronectin and two procollagens, was observed. The focal contact membranes isolated from mouse embryo tibroblasts contained one major protein too (fig. 3, D). Its MW was estimated as 47 kD, i.e. slightly lower than that of chick protein (compare fig. 3, C, D). Discussion We have shown that after treatment of cells with saponin and their removal under the action of shearing force the membranes of focal contacts remain on the substrate. This result confirms the important role of focal contacts in cell-tosubstrate adhesion, since these structures are obviously the most strongly attached regions of lower cell surface. In intact cells the focal contact region of plasma membrane is associated with. dense fibrillar network, forming an adhesive plaque, the continuation of stress tibre [12]. According to electron microscopic data this network is absent in saponin-isolated focal contacts. Using the antibody to vinculin, the protein concentrated at the adhesive plaques, we failed to find it in saponin-isolated contacts. However, it was shown earlier [13] that vinculin is located very closely to the cytoplasmic side of plasma membrane. One may conclude that cytoskeletal elements and even the closest submembranous layer associated with focal contact membrane are removed during the isolation procedure. Since the focal contact membrane devoid of cytoskeletal structures remains firmly attached to the substrate, these structures seem not to be involved in the process of cell adhesion. The method described here allows biochemical study of the cell attachment membrane. The protein composition of saponin-isolated contacts was found to be surprisingly simple. The preparation of focal contact membranes of chick fibroblasts contained only one major cell-derived protein (MW 51 kD). A priori this protein may be a constituent of the saponin-isolated focal contacts themselves or cell-secreted protein adsorbed uniformly on the substrate. The latter hypothesis seems unlikely, since we have not found this protein in cell-conditioned medium. Exp Cell Res 149 (1983)
586 Short notes
In mouse fibroblasts the MW of focal contact membrane major proteins is slightly lower (47 kD) as revealed by gel electrophoresis. This may be due to several reasons. Certainly, 51 and 47 kD proteins may be principally different. But more attractive hypothesis is that they are relative proteins and have the same functions. The difference in the MWs may be the result of evolution divergence. Limited proteolysis of the mouse protein is also not excluded. Peptide mapping analysis is needed to test these possibilities. Aplin et al. [17], using an original cross-linking method, studied the surface proteins of spread BHK cells, which are located just near the substrate. Interestingly, that this fraction of surface proteins was dramatically enriched with a glycoprotein of 47 kD. The antibody to this protein inhibited cell adhesion and even detached the spread cells [ 181. The existence of cellular protein(s) with a MW of 50-55 kD, located closely to the substrate in spread cells, was reported earlier [14]. Quite recently Oesch & Birchmeier [IS] have described the surface protein of chicken tibroblasts with similar MW, called FC-1. The monoclonal antibody to this protein inhibits cell-tosubstrate adhesion and stains preferentially focal contact regions. The relations between 51 and 47 kD proteins, described by us, and cell adhesion proteins, found by others [14, 15, 171, remain to be studied. It seems possible, that these proteins are involved in the processes of cell adhesion and/or membrane association with actin cytoskeleton. We thank Dr S. M. Troyanovsky for advice and help with gel electrophoresis. We also thank Drs A. D. Bershadsky, V. I. Gelfand, I. S. Tint and Professor J. M. Vasiliev for useful discussions and critical reading of the manuscript.
References 1. 2. 3. 4. 5. 6.
Heath, J P & Dunn, G A, J cell sci 29 (1978) 197. Wehland, J, Osborn, M & Weber, K, J cell sci 37 (1979) 257. Geiger, B, Cell 18 (1979) 193. Burridge, K & Feramisco, J R, Cell 19 (1980) 587. Gulp, L A, Murray, B A 8~Rollins, B J, J supramol struct I1 (1979) 401. Badley, R A, Lloyd, C W, Woods, A, Carruthers, L, Allcock, C 8t Rees, D A, Exp cell res 117 (1978) 231. 7. Bershadsky, A D, Gelfand, V I, Svitkina, T M & Tint, I S, Exp cell res 127 (1980) 421. 8. Izzard, C D & Lochner, L R, J cell sci 21 (1976) 129. 9. Spur-r, A R, J ultrastruct res 26 (1969) 31. 10. Laemmli, U K, Nature 227 (1970) 680. 11. Bonner, W M & Laskey, E A, Eur j biochem 46 (1974) 83. 12. Abercrombie, M, Heaysman, J E M & Pegrum, S M, Exp cell res 67 (1971) 359. 13. Geiger, B, Tokuyashi, K T, Dutton, A H & Singer, S J, Proc natl acad sci US 77 (1980) 4127. 14. Lanks, K W & Chin, N W, J cell sci 55 (1982) 137. 15. Oesch, B & Birchmeier, W, Cell 31 (1982) 671. 16. Neyfakh, A A, jr, Tint, J S, Svitkina, T M, Bershadsky, A D & Gelfand, V I, Exp cell res 149 (1983) 387. 17. Aplin, J D, Hughes, R C, Jaffe, C L & Sharon, N, Exp cell res 134 (1981) 488. 18. Hughes, R C, Butters, T D 8~ Aplin, J D, Eur j cell biol 26 (1981) 198. Received June 23, 1983 Printed
in Sweden
Exp Cell Res 149 (1983)
SHORT NOTE Isolation of Focal Contact Membrane Using Saponin A. A. NEYFAKH, Laboratory
of Molecular
JR and T. M. SVITKINA
Biology and Bio-organic Chemistry, University, Moscow 117234, USSR
Moscow
State
The fragments of lower cell surface remained attached to the substrate after incubation of mouse or chick fibroblasts in 0.2% saponin solution and subsequent removal of cells under the action of shearing force. These fragments corresponded exactly to the cellular focal contacts seen by interference reflection microscopy. Ultrastructurally they were membrane fragments with typical three-layered structure. No cytoskeletal components were found in saponin-isolated focal contact membranes either by immunofluorescence or electron microscopy. Only one major cell-derived protein with an apparent molecular weight (MW) of 51 kD (chick embryo fibroblasts) or 47 kD (mouse embryo fibroblasts) remained on the substrate after saponin treatment and removal of cells.
In recent years considerable attention has been focused on the mechanism of cell attachment to the substrate, especially on the structure of focal contacts. Focal contacts are discrete regions of the lower cell surface, located most closely to the substrate, associated with the ends of microfilament bundles [l, 21 and enriched in vinculin [3, 41. In spite of numerous investigations the mechanisms of both membrane-to-substrate attachment and membrane-to-cytoskeleton association in focal contacts remain obscure. Chemical detachment or mechanical removal of cells were used for the purification of the components involved in cell adhesion [5, 61. Here we propose a modification of this approach which allows isolation of pure focal contact membranes . Materials
and Methods
Secondary cultures of mouse or chick embryo tibroblasts were cultivated on coverslips in the 1 : 1 mixture of medium 199 and 0.5 % lactalbumin hydrolysate, supplemented with 10% calf serum. For metabolic labelling chick embryo fibroblasts were grown in roller bottles until subconfluence and then cultivated overnight in serum-free Eagle’s medium, containing 25 @i/ml of 14Camino acids mixture (UVVVR). Proteins secreted by cells were precipitated from this medium with 10% trichloroacetate. For saponin treatment cells were washed with phosphate-buffered saline (PBS) and then incubated for 3-5 min in 0.2% saponin (Merck) in PBS or in cytoskeleton-stabilizing buffer M [7]. Then the cells grown on coverslips were intensively pipetted in the same solution. Roller bottles were vigorously shaken manually. For immunofluorescence staining, specimens were fixed with 4% formaldehyde in PBS. The staining procedure and antibodies to actin and vinculin are described elsewhere [7, 161.The monoclonal antibody to vimentin (clone S47D9) was a generous gift of Dr E B Mechetner. Fluorescence microscopy as well as interference reflection microscopy [8] were performed using Zeiss Photomicroscope III. For transmission electron microscopy (TEM) specimens were fixed with a mixture of 2 % glutaraldehyde and 0.4 % tannic acid in 0.1 M cacodylate buffer (pH 7.2), post-fixed in 1% OsO,, contrasted with uranyl acetate during the ethanol dehydration and embedded in Spurr’s resin [9]. Before ultrathin sectioning the coverslips were dissolved in hydrofluoric acid. Gel electrophoresis was performed according to Laemmli [lo] using 7.5-15 % gradient polyacrylamide slab gels. Labelled proteins were revealed by fluorography [ll]. Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/83 SO3.t?4
Fig. 1. Mouse embryo fibroblast (a, c) before; (b, d) after saponin treatment and pipetting. (a, b) Phase contrast; (c, d) interference reflection. Bar, 20 pm.
Results After incubation of substrate-spread mouse or chick fibroblasts in saponincontaining PBS or buffer M, cells were easily detached from the substrate by means of shearing force (pipetting or shaking). Only small hardly visible cell fragments remained on the glass surface (fig. 1 b). Using interference reflection microscopy [8] these fragments could be easily seen as black strips and spots (fig. 16). Their morphology and distribution were similar to those of focal contact regions in intact cells. The coincidence of cell fragments remaining on the glass and focal contacts was proved by photographing the cell before and after saponin treatment (compare fig. 1 c, 6). Practically all focal contacts remained on the glass, while other regions of the lower cell surface, looking grey or white on the interference reflection picture, were completely removed by sufficiently vigorous pipetting. The fragments remaining on glass, hereafter called ‘saponin-isolated focal contacts’, were examined for the presence of actin and vinculin. The focal contacts of intact cells were brightly stained with anti-vinculin and were found to be associated with the ends of actin bundles. After gentle pipetting, retaining some cells on the substrate, saponin-isolated contacts of removed cells could be Exp Cell Res I49 (19.33)
584 Short notes
Fig. 2. Ultrastructure of saponin-isolated focal contacts of mouse embryo tibroblasts. Note the triplelayered membrane, connected with the substrate by electron-dense bridges. Bottom, Membrane fragment with coated pits. TEM. Bar, 0.1 pm. Fig. 3. Fluorogram of a gel after electrophoresis of “C-labelled proteins. A, Lysate of chick embryo fibroblasts; B, proteins secreted by chick embryo fibroblasts into the medium; C, D, material remaining on the substrate after saponin treatment and removal of chick (C) or mouse (D) tibroblasts. The positions of r4C-labelled marker proteins (Amersham) are indicated.
stained with anti-vinculin. Occasionally the residues of actin bundles were also seen on the substrate. However, after intensive pipetting both actin and vinculin were absent in saponin-isolated focal contacts. As revealed by immunofluorescence staining the protein of intermediate filaments, vimentin, was also absent. The ultrastructure of saponin-isolated focal contacts was investigated by TEM of ultrathin sections perpendicular to the substrate (fig. 2). The substrate surface was identified by the continuous layer of glass-adsorbed serum proteins. Membrane fragments up to several micrometers in size with characteristic threelayered structure were observed closely (about IO-20 nm) to the substrate. Since no other cell-derived material was found on the sections these membranes were evidently saponin-isolated focal contacts. The membranes were connected with the serum protein layer by irregular bridging fibres. There were no structures on Exp Cell Res 149 (1983)
Short notes
585
the inner side of saponin-isolated membranes except occasional coated pits (fig. 2, bottom). In order to study the protein composition of saponin-isolated contacts chick fibroblasts were labelled with 14C-amino acids. The cultures were then treated with saponin and shaken. The material remaining on glass was dissolved in 0.2 % sodium dodecyl sulfate (SDS) and subjected to gel electrophoresis. Only one major protein band, corresponding to MW 51 kD, and a few faint bands were observed on the fluorogram of the gel (fig. 3, C). The addition of the protease inhibitor phenylmethylsulfonylfluoride to all solutions used did not alter the spectrum of labelled polypeptides. The 51 kD protein is not a major protein of intact cells (compare fig. 3, A, C). We have also shown that it is not secreted by fibroblasts into the medium (compare fig. 3, B, C). The secretion of three major polypeptides, which most likely represent fibronectin and two procollagens, was observed. The focal contact membranes isolated from mouse embryo tibroblasts contained one major protein too (fig. 3, D). Its MW was estimated as 47 kD, i.e. slightly lower than that of chick protein (compare fig. 3, C, D). Discussion We have shown that after treatment of cells with saponin and their removal under the action of shearing force the membranes of focal contacts remain on the substrate. This result confirms the important role of focal contacts in cell-tosubstrate adhesion, since these structures are obviously the most strongly attached regions of lower cell surface. In intact cells the focal contact region of plasma membrane is associated with. dense fibrillar network, forming an adhesive plaque, the continuation of stress tibre [12]. According to electron microscopic data this network is absent in saponin-isolated focal contacts. Using the antibody to vinculin, the protein concentrated at the adhesive plaques, we failed to find it in saponin-isolated contacts. However, it was shown earlier [13] that vinculin is located very closely to the cytoplasmic side of plasma membrane. One may conclude that cytoskeletal elements and even the closest submembranous layer associated with focal contact membrane are removed during the isolation procedure. Since the focal contact membrane devoid of cytoskeletal structures remains firmly attached to the substrate, these structures seem not to be involved in the process of cell adhesion. The method described here allows biochemical study of the cell attachment membrane. The protein composition of saponin-isolated contacts was found to be surprisingly simple. The preparation of focal contact membranes of chick fibroblasts contained only one major cell-derived protein (MW 51 kD). A priori this protein may be a constituent of the saponin-isolated focal contacts themselves or cell-secreted protein adsorbed uniformly on the substrate. The latter hypothesis seems unlikely, since we have not found this protein in cell-conditioned medium. Exp Cell Res 149 (1983)
586 Short notes
In mouse fibroblasts the MW of focal contact membrane major proteins is slightly lower (47 kD) as revealed by gel electrophoresis. This may be due to several reasons. Certainly, 51 and 47 kD proteins may be principally different. But more attractive hypothesis is that they are relative proteins and have the same functions. The difference in the MWs may be the result of evolution divergence. Limited proteolysis of the mouse protein is also not excluded. Peptide mapping analysis is needed to test these possibilities. Aplin et al. [17], using an original cross-linking method, studied the surface proteins of spread BHK cells, which are located just near the substrate. Interestingly, that this fraction of surface proteins was dramatically enriched with a glycoprotein of 47 kD. The antibody to this protein inhibited cell adhesion and even detached the spread cells [ 181. The existence of cellular protein(s) with a MW of 50-55 kD, located closely to the substrate in spread cells, was reported earlier [14]. Quite recently Oesch & Birchmeier [IS] have described the surface protein of chicken tibroblasts with similar MW, called FC-1. The monoclonal antibody to this protein inhibits cell-tosubstrate adhesion and stains preferentially focal contact regions. The relations between 51 and 47 kD proteins, described by us, and cell adhesion proteins, found by others [14, 15, 171, remain to be studied. It seems possible, that these proteins are involved in the processes of cell adhesion and/or membrane association with actin cytoskeleton. We thank Dr S. M. Troyanovsky for advice and help with gel electrophoresis. We also thank Drs A. D. Bershadsky, V. I. Gelfand, I. S. Tint and Professor J. M. Vasiliev for useful discussions and critical reading of the manuscript.
References 1. 2. 3. 4. 5. 6.
Heath, J P & Dunn, G A, J cell sci 29 (1978) 197. Wehland, J, Osborn, M & Weber, K, J cell sci 37 (1979) 257. Geiger, B, Cell 18 (1979) 193. Burridge, K & Feramisco, J R, Cell 19 (1980) 587. Gulp, L A, Murray, B A 8~Rollins, B J, J supramol struct I1 (1979) 401. Badley, R A, Lloyd, C W, Woods, A, Carruthers, L, Allcock, C 8t Rees, D A, Exp cell res 117 (1978) 231. 7. Bershadsky, A D, Gelfand, V I, Svitkina, T M & Tint, I S, Exp cell res 127 (1980) 421. 8. Izzard, C D & Lochner, L R, J cell sci 21 (1976) 129. 9. Spur-r, A R, J ultrastruct res 26 (1969) 31. 10. Laemmli, U K, Nature 227 (1970) 680. 11. Bonner, W M & Laskey, E A, Eur j biochem 46 (1974) 83. 12. Abercrombie, M, Heaysman, J E M & Pegrum, S M, Exp cell res 67 (1971) 359. 13. Geiger, B, Tokuyashi, K T, Dutton, A H & Singer, S J, Proc natl acad sci US 77 (1980) 4127. 14. Lanks, K W & Chin, N W, J cell sci 55 (1982) 137. 15. Oesch, B & Birchmeier, W, Cell 31 (1982) 671. 16. Neyfakh, A A, jr, Tint, J S, Svitkina, T M, Bershadsky, A D & Gelfand, V I, Exp cell res 149 (1983) 387. 17. Aplin, J D, Hughes, R C, Jaffe, C L & Sharon, N, Exp cell res 134 (1981) 488. 18. Hughes, R C, Butters, T D 8~ Aplin, J D, Eur j cell biol 26 (1981) 198. Received June 23, 1983 Printed
in Sweden
Exp Cell Res 149 (1983)