Proc. Nat. Acad. Sci. USA
Vol. 73, No. 2, pp. 577-581, February 1976
Cell Biology
Mechanisms of adhesion among cells from neural tissues of the chick embryo (cell-cell binding/brain and retinal cells/cell surface proteins/proteolytic activation)
URS RUTISHAUSER, JEAN-PAUL THIERY, ROBERT BRACKENBURY, BEN-AMI SELA, AND GERALD M. EDELMAN The Rockefeller University, New York, N.Y. 10021
Contributed by Gerald M. Edelman, December 9,1975
the overall control of adhesion during the development of these two tissues is discussed in terms of this model.
In order to analyze the molecular mechaABSTRACT nisms of cell adhesion during development, proteins on the surface of chick embryonic neural cells were compared with proteins released after placing these cells in culture. One of the components released into culture, F1 (molecular weight, M, 140,000), was derived by proteolytic cleavage of a cell surface precursor with a molecular weight of at least 240,000. Another proteinj F2, recovered from culture as a dimer (Mr 110,000), appeared to be a product of limited proteolytic cleavage of Fl. Cells in retina tissue possessed a surface protein of Mr 150,000 that also appeared to be derived by limited proteolytic cleavage of the cell surface precursor. Antibodies to F2 interacted with determinants on the cell surface protein of Mr 150,000, and specifically prevented homologous and heterologous binding among dissociated retinal and brain cells. In contrast, antibodies to F1 failed to prevent cell-cell adhesion and did not crossreact with F2. These data suggest that the cell surface protein of Mr 150,000 generated by limited proteolysis is involved in adhesion of both retinal and brain cells. Cell-cell binding of both retinal and brain cells varied as a function of developmental age and brain cells acquired their binding properties at an earlier time than retinal cells. Similar results were obtained in experiments on the binding of retinal and brain cells of different ages to nylon fibers coated with antibodies to F2. The results of the molecular and cellular experiments are incorporated in a model for cell adhesion invoking both proteolytic activation and modulation of cell surface ligands.
MATERIALS AND METHODS Preparation of Tissues and Cells. Neural retina and brain tissues from white leghorn chick embryos were treated with 0.5% trypsin (1-250, Nutritional Biochemicals Corp.) in phosphate-buffered saline pH 7.3, for 20 min at 370, washed three times, resuspended in medium (Dulbecco's Modified Eagle Medium, Grand Island Biological Co., containing glutamine, penicillin, streptomycin, and 20 ,g/ml of DNase I), and then dispersed into single cells by pipetting. The dissociated cells were allowed to recover for 12-24 hr at 37° either in suspension cultures without serum, calcium or magnesium, or as monolayers in medium with 5% fetal calf serum. With suspension cultures the protease inhibitor Trasylol (20 units/ml, Calbiochem) was added. Suspension cultures yielded mostly single cells with a viability of 8090%.
Isolation of Proteins F1 and F2 from Retina. Retinas from 10-day embryos were cultured either as intact tissue in medium with Trasylol, or as cell monolayers in medium with 5% serum. Supernatants from cultures of intact tissue (TCS) were collected after 24 hr and centrifuged to remove debris. Cells in monolayer cultures were washed and incubated for 24-48 hr in medium without serum but with Trasylol, after which the monolayer culture supernatant (MCS) was collected and centrifuged. After a 30-fold concentration by ultrafiltration through Amicon PM-10 membranes, MCS and TCS from 400 retinas were passed through DE-52 DEAE-cellulose (Whatman) columns in 0.01 M Tris-HCl-0.4 M KCl, pH 7.2, to remove nucleic acids, and fractionated on a 0.5 X 10 cm column of DE-52 in 0.01 M Tris, pH 7.2, with a linear gradient from 0.01 to 0.4 M KCl. Fractions containing the F1 or F2 proteins (see Fig. 1) were identified by analytical polyacrylamide gel electrophoresis, pooled, and further separated by preparative gel electrophoresis on 1 cm X 10 cm cylindrical gels of 6.5% acrylamide in Tris-glycine buffer (6). F1 and F2 were located by scanning the gels at 280 nm with a Gilford spectrophotometer and eluted from the appropriate gel slice by electrophoresis (7). The yield of F1 from MCS was about 250 Mg. The yield of F2 from TCS varied from 50 to
Cell adhesion has attracted the attention of biologists in a variety of fields because it reflects some of the most fundamental aspects of metazoan physiology. Several attempts have been made to analyze cell adhesion, but as yet its physical basis remains unknown. The main conclusion that can be drawn from these studies (1-5) is that adhesion is a complex phenomenon that in some way includes molecular ligation of cell surfaces. In view of this complexity, an experimental approach requires an analysis of (a) the structure of molecules expressed at and released from the cell surface; (b) the specific interactions among these molecules; (c) the relationship of the properties of these molecules to cell-cell binding in kinetically defined assays; and (d) the relationship of this binding to tissue formation. In this paper, we describe the application of this approach to the study of adhesion among dissociated retinal and brain cells from chick embryos. These studies suggest that proteolytic alteration of a major cell surface protein results in the appearance of determinants required for the initial formation of bonds between both retinal and brain cells. A model of the activation and ligation steps has been formulated and
150,gg.
Preparation of Antibodies. Rabbits were immunized at monthly intervals with 100 ,4g of purified F1 or F2 in Freund's adjuvant. The immunoglobulin fraction from the antisera was used in all experiments. To detect F1 or F2 on the cell surface membrane, cells were incubated with antibody labeled with fluorescein (8) or 125I (9) (50-1000 lg/ml,
Abbreviations: CAM, cell adhesion molecule; MCS, monolayer culture supernatant; TCS, tissue culture supernatant; Mr, molecular weight.
577
578
Proc. Nat. Acad. Sci. USA 73 (1976)
Cell Biology: Rutishauser et al. 0.5
0.4
0.4
0.3
0.3 ._,
02
0.2
0.1
0.1
y>u
0.6 04 0.2 0
OD cxi
Relative mobility
FIG. 1. Isolation of the F1 and F2 proteins from culture supernatants of retina cells. (a) Ion-exchange chromatography of monolayer culture supernatant (MCS) on DE-52. F1 is contained in fraction A. (b) Scan of preparative gel electrophoresis of fraction A on 6.5% polyacrylamide gels in Tris-glycine. Pure F1 was eluted from the region of the gel indicated by the hatched peak. (c) Ionexchange chromatography of tissue culture supernatant (TCS) on DE-52. F2 is contained in fraction B. (d) Preparative gel electrophoresis of fraction B. Pure F2 was eluted from the indicated region of the gel.
15 min, 370), washed, and the
presence of bound antibody by fluorescence microscopy or by measurement of radioactivity. Immunioprecipitation of Nonidet P40 Extracts of Labeled Cell Surface Proteins (10). Cell surface proteins in retinal tissue, cell monolayers, and on suspension culture cells were labeled with 125I by the lactoperoxidase procedure. The proteins were then extracted with 0.5% Nonidet P-40, immunoprecipitated by incubation with anti-Fl or anti-F2 followed by goat-anti-rabbit antibody, solubilized in 2% sodium dodecyl sulfate-2% 2-mercaptoethahol, and fractionated by electrophoresis on 6.5% sodium dodecyl sulfate polyacrylamide gels (11). The fractionated proteins were detected by measurement of radioactivity in 2 mm gel slices. Cell-Cell Binding Assay. This assay is based on the immobilization of cells on nylon fibers or culture dishes (12) and a subsequent analysis of the ability of cells in suspension to bind to the immobilized cells (13). To immobilize the cells, fibers or dishes were coated with waxbean agglutinin
was- determined
(14) and incubated with 107 cells in medium. After washing, anti-waxbean agglutinin (200 ,gg/ml) was added to prevent further binding of cells by the lectin, and the immobilized cells were then oscillated on an Eberbach reciprocal shaker (70 rpm, 30 min, 250) with 2 X 106 cells suspended in medium. The cells in suspension had been internally labeled by incubation with 20 ,ug/ml of fluorescein diacetate (15), so that after washing, the number of cell-bound cells could be counted using fluorescence microscopy. Cell-Fiber Binding Assay. Details of this assay have been published elsewhere (12). Briefly, nylon fibers were incubated with a 0.5 mg/ml solution of antibody and then incubated for 30 min at 23' with 4 X 106 cells suspended in 4 ml of medium. Unbound cells were washed away and the fiberbound cells were counted at 125X magnification. RESULTS Several experimental procedures were coordinated in order to examine the molecular mechanism of cell adhesion: (1) The isolation of proteins released by cultured retina cells, and the use of antibodies to these proteins to identify those present on the cell surface. (2) The physical and immunological characterization of two proteins, Fl and F2, with an analysis of the form in which they exist on the cell surface. (3) A quantitative analysis of initial adhesion among individual retina and brain cells, and the implication of F2 in this process. (4) An analysis at different developmental ages of the interaction between neural cells and nylon fibers coated with antibodies to F2. Isolation and Characterization of Proteins from Retina Culture Supernatants. Fractionation of TCS and MCS by ion exchange chromatography and gel electrophoresis yielded a number of purified proteins (Fig. 1). Antibodies were prepared to these proteins and immunofluorescence microscopy indicated that two of them existed in some form on the cell surface. One of these, Fl, was isolated from MCS (Fig. la and b), and the other, F2, was isolated from TCS (Fig. lc and d). The molecular weights of Fl and F2 under nondissociating conditions were estimated by gel exclusion chromatography on Sephadex G-200 (16) to be 140,000 and 110,000, respectively. Gel electrophoresis of F1 in sodium dodecyl sulfate with 2-mercaptoethanol (11) gave a single component with a molecular weight (Mr) of 140,000. F2 on the same gels migrated as a single component of Mr 55,000. This suggested that under nondissociating conditions F2 exists as a dimer composed of two identical chains. Immunodiffusion studies with antibodies produced against F1 (anti-Fl) or F2 (anti-F2) indicated that although F1 and F2 shared some antigenic determinants, these two antisera were primarily directed against determinants unique to each protein. As shown in Fig. 2, anti-Fl reacted strongly with Fl but not detectably with F2, whereas, antiF2 reacted strongly with F2 and weakly with Fl. The relationship between Fl and F2 was more clearly indicated by two observations: (1) When released by cells in the absence of the protease inhibitor Trasylol, F1 purified under nondissociating conditions often migrated as several components in sodium dodecyl sulfate-gel electrophoresis. Such Fl molecules also dissociated into several pieces upon standing in physiological medium; the major component had the same electrophoretic mobility, molecular weight, and antigenic determinants as F2. (2) When intact F1 was partially degraded by trypsin (25 ,g/ml, 30 min, 370), a num-
Proc. Nat. Acad. Sci. USA 73 (1976)
Cell Biology: Rutishauser et al.
Anti - F 1
F1
F2 Anti - F 2
FIG. 2. Immunodiffusion analysis of F1 and F2 using antibodies to the purified proteins.
ber of fragments were produced, including F2. These results, along with the immunological and molecular weight data, indicate that monomeric F1 contains a protease-sensitive region that, when cleaved under nondenaturing conditions yields F2. Anti-Fl and anti-F2, although produced against purified proteins, reacted with a number of minor components in MCS and TCS. The proteins from MCS reacted more strongly with anti-Fl, and those from TCS reacted more strongly with anti-F2. All of these proteins shared antigenic determinants with F1 and/or F2. The data strongly suggest that these molecules are derived from the same molecule, and also confirm the specificity of the antisera. Identification and Characterization of F1 and F2 on the Cell Surface. Antibodies to F1 and F2 were used to study these and structurally related proteins on the surface of cells grown in suspension, monolayers, or intact tissue. In all three cases, fluorescein-labeled anti-Fl and anti-F2 stained retinal cells brain cells. These antibodies also stained chick embryo fibroblasts. They did not, however, stain chick embryo thymocytes or erythrocytes. Staining with anti-F2 was stronger than with anti-Fl, and on nearly all cells the fluorescence was distributed in small patches over the entire membrane. For correlation with cell-cell and cell-fiber binding experiments (see below), the amount of anti-Fl or anti-F2 bound by retinal and brain cells at particular stages of development was estimated in suspension using 125I-labeled antibody. Under saturating conditions (1 mg/ml of antibody), retinal cells from 8- and 13-day embryos and brain cells
from 6- and 10-day-old embryos bound on the average 2 to 4 X 105 anti-F2 molecules and 0.6 to 1.0 X 105 anti-Fl molecules per cell. As expected from these data on intact cells, anti-Fl and anti-F2 also precipitated 125I-labeled cell surface proteins extracted from retina cells (Fig. 3). When cells from monolayers were used, similar amounts of protein were precipitated by either antibody. With both antibodies this included a major component with a molecular weight in sodium dodecyl sulfate of 240,000, and minor components with molecular weights of 265,000, 175,000, and 65,000. When nondissociated retinal tissue or cells from suspension culture were extracted, however, most of the material precipitated by the same antibodies had a molecular weight in sodium dodecyl sulfate of 150,000, with a minor component of Mr 175,000; in this experiment, the anti-F2 precipitated about four times more protein than anti-Fl. Immunoglobulin from unimmunized rabbits did not precipitate significant amounts of protein. The data suggest that F1 and F2 are soluble cleavage products of a cell surface polypeptide, with a molecular weight of 240,000, and that in tissue or in suspension cultures the majority of these molecules are converted to polypeptides with a molecular weight of 150,000. In addition, the specific precipitation of components with molecular weights of 265,000 and 175,000 suggests that another cleavage, removing a fragment of Mr 25,000 from the membrane-bound molecules, may also have occurred. Binding among Retinal and Brain Cells from Embryos of Different Ages. In Table 1 are summarized the binding frequencies observed among trypsin-dissociated neural cells in the cell-cell binding assay and the effect of anti-F2 on this binding. High levels of binding were obtained between pairs of 8-day retinal cells, pairs of 6-day brain cells, and between pairs consisting of a 8-day retinal cell and a 6-day brain cell. If, however, the trypsinized cells were not allowed to recover in suspension cultures, no cell-cell binding occurred. Full recovery was achieved in 6-12 hr. In contrast, even fully recovered 13-day retinal cells and 10-day brain cells bound poorly to themselves and to each other. If 8-day retinal cells and 6-day brain cells were incubated with anti-F2 (1 mg/ml, 15 min, 250) prior to the assay, binding was inhibited by 75-90%. In contrast, preincubation with anti-Fl did not affect binding with either cell type. Binding of Cells to Fibers Coated with Anti-F2. The
Molecular weight (x 10-3) 50
Table 1. Binding among retinal and brain cells from embryos of different ages
250 200 50 100
1000I
579
a
Cell-celli binding between: * Cell A Cell B
5001
a.
Cell-bound
cellst
Binding in
presence of
anti-F2t
0
b
R8 B6 R8
500
RI3
B10
0
02
0.6
1.0
R8
Relative mobility
FIG. 3. Sodium dodecyl sulfate-gel electrophoresis of 125I-labeled retinal cell surface proteins precipitated by anti-F2. The same proteins were precipitated by anti-Fl. (a) Proteins from trypsin-dissociated cells grown in monolayers. (b) Proteins from cells in undissociated retinal tissue. Molecular weights in sodium dodecyl sulfate-2-mercaptoethanol were assigned relative to the migration of 1311-labeled spectrin, f3-galactosidase, and immunoglobulin heavy chains in the same 6.5% polyacrylamide gels.
R8 *
Rs B6
B6
RI3 B10 RI3
B10
423 413 390 122 8 175 41
37 32 41
R8 and R13 are cells from retinas of 8- and 13-day-old chick embryos; B6 and B1o are cells from brains of 6- and 10-day-old em-
bryos.
t Expressed as number of B cells bound to 1 mm2 of A cell monolayer.
t Both Cell A and Cell B were incubated for 15 min at 250 with antibodies to F2 (1 mg/ml) prior to cell-cell binding.
580
Proc. Nat. Acad. Sci. USA 73 (1976)
Cell Biology: Rutishauser et al.
Cleavage at B
w
1000
0
mE
U800
CL
E E
0' a)
i5 600 (
0
Mr 140,000 Cleavage at A
a)
400
-o? 1
A
u-
*
200
C)
0 100 0E 0
c
30
0
Mr
1 50,000
10 -)
_-..---anti-F2 3
4
6
8
10
12
14
r
559000 L. F2 dimer
Fetal days
FIG. 4. Age dependence of cell binding to fibers coated with anti-F2. (a) Binding of retinal and brain cells to fibers as a function of embryonic age. The vertical axis (FBC/cm) indicates the number of cells bound along one edge of a one cm fiber segment. (b) Increase in the number of cells obtained by dissociation of retinal or brain tissue as a function of embryonic age.
ability of cells to bind to nylon fibers coated with anti-F2 also depended upon the age of the embryo, and different profiles were obtained for retinal and brain cells (Fig. 4a). Cells from both tissues of a 4-day embryo bound poorly to the fibers. The binding of brain cells increased to a maximum by 6 4dys and then decreased. Retinal cells did not bind until 5-6 days, reached maximal binding at 8-9 days, and then decreased in their binding to low levels by the 13th day. Binding of brain and retinal cells was inhibited 80-90% by preincubation of the anti-F2 fiber with F2 (20.ug/ml). In contrast to these results, binding of brain and retinal cells to fibers coated with the lectin concanavalin A or antibodies prepared against chick liver mlls that also bind to neural cells did not vary more than about 30% as a function of embryonic age. As with cell-cell binding, cells that were not allowed to recover from trypsinization did not bind to anti-F2 fibers. Maximum levels of binding were obtained after a recovery period of only 2-4 hr, in contrast to the cell-cell binding, which required 6-12 hr. In Fig. 4 is also shown a graph of the numbers of cells in a retina or brain as a function of embryonic age. Although these numbers may reflect a complex pattern of cell division, death, and migration, it is striking that, for both tissues, the rate of increase in cell number was temporally related to the ability of their cells to bind to other cells or to anti-F2 fibers. DISCUSSION
These studies of F1 and F2 and related proteins on brain and retinal cell surfaces can be interpreted in terms of the model shown in Fig. 5. This discussion, therefore, emphasizes the bases and the consequences of this model. The data on initial binding events among individual brain and retinal cells suggest that the molecular mechanism of cell adhesion is the same in these two tissues. Brain and retinal cells can bind to each other as well as to themselves, and in all cases this binding can be inhibited by anti-F2. The different temporal patterns displayed in the cell-cell binding
Cell-cell binding FIG. 5. Proposed schematic model for the formation of cellcell bonds by a cell adhesion molecule (CAM). Both the activation of CAM from proCAM and the production of soluble fragments F1, F2, and F3 from proCAM are shown. The F3 component has not been isolated and may be further degraded. Of the three possible mechanisms for CAM-mediated eell-cell binding, only the symmetrical CAM-CAM bond is shown. A description of these mechanisms as well as the assignment of cleavages and molecular weights is contained in the text. The dashed arrow indicates the blockage of cell-cell binding by reaction of anti-F2 with CAM. appear to be under control of factors other than simply the display of aggregation-promoting molecules unique to each tissue (17). In view of the specific inhibition of cell-cell binding by antibodies to F2, we suggest that structures on a cell surface molecule possessing antigenic determinants in common with F2 participate in the binding rNotion. This conclusion is further supported by the temporal correlation between the ability of a cell to bind to a fiber coated with anti-F2 and its ability to bind to another cell. The problem is therefore to relate the antigenic determinants on F2 to a cell surface molecule. In the model it is proposed that these determinants are present on a cell adhesion molecule (CAM), which contains F2 as an integral portion of its single polypeptide chain. CAM, which is found on cells in tissue or cultured in suspension, is generated by proteolytic cleavage of a precursor molecule, proCAM, which is found on the surface of retinal cells grown in monolayers. The model, therefore, depicts proCAM as having at least two regions that are sensitive to proteolytic cleavage. One of these cleavage sites, called B in Fig. 5, involves the release of fragment F1 into culture medium. The other, called the A site, would account for two cleavages; (1) The formation of the F2 subunit from F1, and (2) The conversion of proCAM into CAM. In solution, the A site cleavage releases the F2 subunit that subsequently forms a dimer. The same cleavage of proCAM would produce the cell surface molecule CAM, which
experiments, therefore,
Cell Biology: Rutishauser et al. carries the antigenic determinants involved in cell-cell binding.
The ability of anti-F2 but not anti-Fl to inhibit cell-cell binding is consistent with the proposed model. Anti-F2 reacts most strongly with the F2-related determinants on CAM and, therefore, prevents CAM-mediated cell-cell binding. On the other hand, anti-Fl antibodies do not react with F2 antigenic determinants, and would not necessarily be expected to block adhesion. Assuming that CAM participates directly in cell adhesion, it still remains to be specified how cell-cell bonds are formed. There are three main possibilities: (1) CAMs on different cells bind directly to each other. (2) CAMs on different cells are bridged by an as yet unidentified molecule. (3) CAMs on one cell bind to an unidentified receptor on another cell. At this time, we favor the first mechanism because of its simplicity and because F2 exists as a dimer in solution. It should be noted, however, that it has not been proven that binding can occur between two CAMs. The activation of proCAM by proteolysis raises the possibility that cell adhesion is controlled by mechanisms similar to those observed in blood clotting and complement fixation. The presence of many Fl- and F2-related fragments in MCS and TCS suggests that the cleavage mechanisms could be more complex than shown in Fig. 5. Furthermore, breakage of cell-cell adhesions might involve cleavage at site B, with the consequent release of F2 dimers. The correlation between the binding of cells to fibers coated with anti-F2 and their ability to bind to other cells suggests that another mechanism may also control cell adhesion. The amount of soluble anti-F2 and anti-Fl that binds to the surface of cells from brain and retina did not change significantly with embryonic development. At first glance, this observation would seem to be inconsistent with the temporal variation observed in the cell-cell and cell-fiber assays. It has been shown, however, that the distribution or mobility of molecules at the cell surface can affect their ability to form cell-cell and cell-fiber bonds (13, 18). In this respect it is noteworthy that the mobility of cell surface molecules can be modulated by a network of cytoplasmic microtubules and microfilaments (19). While it has not been established whether such a network is involved in morphogenesis, it is clear that receptor properties such as mobility or distribution must be considered in analyzing cell adhesion. It is particularly striking that the temporal increase in cell numbers in brain or retinal tissues was correlated with increases in cell-cell and cell-fiber binding. This suggests that changes in cell surface properties coordinated with the cell cycle may also affect cell adhesion. In view of its molecular weight and presence on the cell surface, proCAM may resemble the transformation-sensitive protein described by a number of laboratories (see ref. 20). In fact, it has been suggested that this protein, which has a molecular weight of 250,000 and spans the cell membrane (21), might in some way be involved with modulation of re-
Proc. Nat. Acad. Sci. USA 73 (1976)
581
ceptor mobility, growth control, and agglutination reactions (22). The two molecules appear to differ significantly in the size of their cleavage products, however, and we have not been able to detect any immunological crossreaction between them. Nevertheless, the information available so far is not sufficient to rule out the possibility that they are related. In any event, it will be important to determine whether proCAM spans the cell membrane and is associated with intracellular networks of microtubules and microfilaments comprising a cell surface modulating assembly (23). We are grateful to Ms. Mildred Decker and Ms. Helvi Hjelt for excellent technical assistance. This work was supported by U.S. Public Health Service Grants HD 09635, Al 11378, Al 09273, AM 04256. J.-P.T. is a fellow of the International Agency for Research on Cancer, Lyon, France, R.B. is a fellow of the Jane Coffin Childs Memorial Fund for Medical Research, and B.S. is supported by a fellowship from the Damon Runyon-Walter Winchell Cancer Fund. 1. Weiss, L. (1968) The Cell Periphery, Metastasis and Other Contact Phenomena, eds. Neuberger, A. & Tatum, E. L. (American Elsevier, New York). 2. Moscona, A. A. (1973) in Cell Biology in Medicine, ed. Bittar, E. E. (Wiley-Interscience, New York), pp. 571-591. 3. Steinberg, M. S. (1970) J. Exp. Zool. 173,395-434. 4. Curtis, A. S. G. (1967) The Cell Surface (Academic Press, New York). 5. Balsamo, J. & Lilien, J. (1974) Nature 251, 522-524. 6. Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-436. 7. Stephens, R. E. (1975) Anal. Biochem. 65,369-379. 8. Cebra, J. J. & Goldstein, G. (1965) J. Immunol. 95, 230-245. 9. McConahey, P. J. & Dixon, F. J. (1966) Int. Arch. Allergy Appl. Immunol. 29, 185-189. 10. Henning, R., Milner, R. J., Reske, K., Cunningham, B. A. & Edelman, G. M. (1976) Proc. Nat. Acad. Sci. USA 73, 118122. 11. Laemmli, U.K. (1970) Nature 227,680-685. 12. Edelman, G. M. & Rutishauser, U. (1974) in Methods in Enzymology, eds. Jakoby, W. & Wilchek, M. (Academic Press, New York), pp. 195-225. 13. Rutishauser, U. & Sachs, L. (1974) Proc. Nat. Acad. Sci. USA
71,2456-2460. 14. Sela, B., Lis, H., Sharon, N. & Sachs, L. (1973) Biochim. Biophys. Acta 310, 273-277. 15. Bodmer, W. F., Tripp, M. & Bodmer, J. (1967) in Histocompatibility Testing, 1967 (Munksgaard, Copenhagen), pp. 341-350. 16. Ackers, G. A. (1970) Adv. Protein Chem. 24,343-446. 17. Rutishauser, U., Yang, D., Thiery, J.-P. & Edelman, G. M. (1975) in Proceedings of the 1975 Conference on Development Biology, ICN-UCLA Symposia, in press. 18. Rutishauser, U. & Sachs, L. (1975) J. Cell Biol. 66,76-85. 19. Edelman, G. M., Yahara, I. & Wang, J. L. (1973) Proc. Nat. Acad. Sci. USA 70,1442-1446. 20. Hynes, R. 0. (1974) Cell 1, 147-155. 21. Hunt, R. C. & Brown, J. C. (1975) J. Mol. Biol. 97, 413-422. 22. Yamada, K. M., Yamada, S. S. & Pastan, I. (1975) Proc. Nat. Acad. Sci. USA 72,3158-3162. 23. Edelman, G. M., Wang, J. L. & Yahara, I. (1975) "Cell motility," Cold Spring Harbor Symp. Quant. Biol., in press.
Vol. 73, No. 2, pp. 577-581, February 1976
Cell Biology
Mechanisms of adhesion among cells from neural tissues of the chick embryo (cell-cell binding/brain and retinal cells/cell surface proteins/proteolytic activation)
URS RUTISHAUSER, JEAN-PAUL THIERY, ROBERT BRACKENBURY, BEN-AMI SELA, AND GERALD M. EDELMAN The Rockefeller University, New York, N.Y. 10021
Contributed by Gerald M. Edelman, December 9,1975
the overall control of adhesion during the development of these two tissues is discussed in terms of this model.
In order to analyze the molecular mechaABSTRACT nisms of cell adhesion during development, proteins on the surface of chick embryonic neural cells were compared with proteins released after placing these cells in culture. One of the components released into culture, F1 (molecular weight, M, 140,000), was derived by proteolytic cleavage of a cell surface precursor with a molecular weight of at least 240,000. Another proteinj F2, recovered from culture as a dimer (Mr 110,000), appeared to be a product of limited proteolytic cleavage of Fl. Cells in retina tissue possessed a surface protein of Mr 150,000 that also appeared to be derived by limited proteolytic cleavage of the cell surface precursor. Antibodies to F2 interacted with determinants on the cell surface protein of Mr 150,000, and specifically prevented homologous and heterologous binding among dissociated retinal and brain cells. In contrast, antibodies to F1 failed to prevent cell-cell adhesion and did not crossreact with F2. These data suggest that the cell surface protein of Mr 150,000 generated by limited proteolysis is involved in adhesion of both retinal and brain cells. Cell-cell binding of both retinal and brain cells varied as a function of developmental age and brain cells acquired their binding properties at an earlier time than retinal cells. Similar results were obtained in experiments on the binding of retinal and brain cells of different ages to nylon fibers coated with antibodies to F2. The results of the molecular and cellular experiments are incorporated in a model for cell adhesion invoking both proteolytic activation and modulation of cell surface ligands.
MATERIALS AND METHODS Preparation of Tissues and Cells. Neural retina and brain tissues from white leghorn chick embryos were treated with 0.5% trypsin (1-250, Nutritional Biochemicals Corp.) in phosphate-buffered saline pH 7.3, for 20 min at 370, washed three times, resuspended in medium (Dulbecco's Modified Eagle Medium, Grand Island Biological Co., containing glutamine, penicillin, streptomycin, and 20 ,g/ml of DNase I), and then dispersed into single cells by pipetting. The dissociated cells were allowed to recover for 12-24 hr at 37° either in suspension cultures without serum, calcium or magnesium, or as monolayers in medium with 5% fetal calf serum. With suspension cultures the protease inhibitor Trasylol (20 units/ml, Calbiochem) was added. Suspension cultures yielded mostly single cells with a viability of 8090%.
Isolation of Proteins F1 and F2 from Retina. Retinas from 10-day embryos were cultured either as intact tissue in medium with Trasylol, or as cell monolayers in medium with 5% serum. Supernatants from cultures of intact tissue (TCS) were collected after 24 hr and centrifuged to remove debris. Cells in monolayer cultures were washed and incubated for 24-48 hr in medium without serum but with Trasylol, after which the monolayer culture supernatant (MCS) was collected and centrifuged. After a 30-fold concentration by ultrafiltration through Amicon PM-10 membranes, MCS and TCS from 400 retinas were passed through DE-52 DEAE-cellulose (Whatman) columns in 0.01 M Tris-HCl-0.4 M KCl, pH 7.2, to remove nucleic acids, and fractionated on a 0.5 X 10 cm column of DE-52 in 0.01 M Tris, pH 7.2, with a linear gradient from 0.01 to 0.4 M KCl. Fractions containing the F1 or F2 proteins (see Fig. 1) were identified by analytical polyacrylamide gel electrophoresis, pooled, and further separated by preparative gel electrophoresis on 1 cm X 10 cm cylindrical gels of 6.5% acrylamide in Tris-glycine buffer (6). F1 and F2 were located by scanning the gels at 280 nm with a Gilford spectrophotometer and eluted from the appropriate gel slice by electrophoresis (7). The yield of F1 from MCS was about 250 Mg. The yield of F2 from TCS varied from 50 to
Cell adhesion has attracted the attention of biologists in a variety of fields because it reflects some of the most fundamental aspects of metazoan physiology. Several attempts have been made to analyze cell adhesion, but as yet its physical basis remains unknown. The main conclusion that can be drawn from these studies (1-5) is that adhesion is a complex phenomenon that in some way includes molecular ligation of cell surfaces. In view of this complexity, an experimental approach requires an analysis of (a) the structure of molecules expressed at and released from the cell surface; (b) the specific interactions among these molecules; (c) the relationship of the properties of these molecules to cell-cell binding in kinetically defined assays; and (d) the relationship of this binding to tissue formation. In this paper, we describe the application of this approach to the study of adhesion among dissociated retinal and brain cells from chick embryos. These studies suggest that proteolytic alteration of a major cell surface protein results in the appearance of determinants required for the initial formation of bonds between both retinal and brain cells. A model of the activation and ligation steps has been formulated and
150,gg.
Preparation of Antibodies. Rabbits were immunized at monthly intervals with 100 ,4g of purified F1 or F2 in Freund's adjuvant. The immunoglobulin fraction from the antisera was used in all experiments. To detect F1 or F2 on the cell surface membrane, cells were incubated with antibody labeled with fluorescein (8) or 125I (9) (50-1000 lg/ml,
Abbreviations: CAM, cell adhesion molecule; MCS, monolayer culture supernatant; TCS, tissue culture supernatant; Mr, molecular weight.
577
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Proc. Nat. Acad. Sci. USA 73 (1976)
Cell Biology: Rutishauser et al. 0.5
0.4
0.4
0.3
0.3 ._,
02
0.2
0.1
0.1
y>u
0.6 04 0.2 0
OD cxi
Relative mobility
FIG. 1. Isolation of the F1 and F2 proteins from culture supernatants of retina cells. (a) Ion-exchange chromatography of monolayer culture supernatant (MCS) on DE-52. F1 is contained in fraction A. (b) Scan of preparative gel electrophoresis of fraction A on 6.5% polyacrylamide gels in Tris-glycine. Pure F1 was eluted from the region of the gel indicated by the hatched peak. (c) Ionexchange chromatography of tissue culture supernatant (TCS) on DE-52. F2 is contained in fraction B. (d) Preparative gel electrophoresis of fraction B. Pure F2 was eluted from the indicated region of the gel.
15 min, 370), washed, and the
presence of bound antibody by fluorescence microscopy or by measurement of radioactivity. Immunioprecipitation of Nonidet P40 Extracts of Labeled Cell Surface Proteins (10). Cell surface proteins in retinal tissue, cell monolayers, and on suspension culture cells were labeled with 125I by the lactoperoxidase procedure. The proteins were then extracted with 0.5% Nonidet P-40, immunoprecipitated by incubation with anti-Fl or anti-F2 followed by goat-anti-rabbit antibody, solubilized in 2% sodium dodecyl sulfate-2% 2-mercaptoethahol, and fractionated by electrophoresis on 6.5% sodium dodecyl sulfate polyacrylamide gels (11). The fractionated proteins were detected by measurement of radioactivity in 2 mm gel slices. Cell-Cell Binding Assay. This assay is based on the immobilization of cells on nylon fibers or culture dishes (12) and a subsequent analysis of the ability of cells in suspension to bind to the immobilized cells (13). To immobilize the cells, fibers or dishes were coated with waxbean agglutinin
was- determined
(14) and incubated with 107 cells in medium. After washing, anti-waxbean agglutinin (200 ,gg/ml) was added to prevent further binding of cells by the lectin, and the immobilized cells were then oscillated on an Eberbach reciprocal shaker (70 rpm, 30 min, 250) with 2 X 106 cells suspended in medium. The cells in suspension had been internally labeled by incubation with 20 ,ug/ml of fluorescein diacetate (15), so that after washing, the number of cell-bound cells could be counted using fluorescence microscopy. Cell-Fiber Binding Assay. Details of this assay have been published elsewhere (12). Briefly, nylon fibers were incubated with a 0.5 mg/ml solution of antibody and then incubated for 30 min at 23' with 4 X 106 cells suspended in 4 ml of medium. Unbound cells were washed away and the fiberbound cells were counted at 125X magnification. RESULTS Several experimental procedures were coordinated in order to examine the molecular mechanism of cell adhesion: (1) The isolation of proteins released by cultured retina cells, and the use of antibodies to these proteins to identify those present on the cell surface. (2) The physical and immunological characterization of two proteins, Fl and F2, with an analysis of the form in which they exist on the cell surface. (3) A quantitative analysis of initial adhesion among individual retina and brain cells, and the implication of F2 in this process. (4) An analysis at different developmental ages of the interaction between neural cells and nylon fibers coated with antibodies to F2. Isolation and Characterization of Proteins from Retina Culture Supernatants. Fractionation of TCS and MCS by ion exchange chromatography and gel electrophoresis yielded a number of purified proteins (Fig. 1). Antibodies were prepared to these proteins and immunofluorescence microscopy indicated that two of them existed in some form on the cell surface. One of these, Fl, was isolated from MCS (Fig. la and b), and the other, F2, was isolated from TCS (Fig. lc and d). The molecular weights of Fl and F2 under nondissociating conditions were estimated by gel exclusion chromatography on Sephadex G-200 (16) to be 140,000 and 110,000, respectively. Gel electrophoresis of F1 in sodium dodecyl sulfate with 2-mercaptoethanol (11) gave a single component with a molecular weight (Mr) of 140,000. F2 on the same gels migrated as a single component of Mr 55,000. This suggested that under nondissociating conditions F2 exists as a dimer composed of two identical chains. Immunodiffusion studies with antibodies produced against F1 (anti-Fl) or F2 (anti-F2) indicated that although F1 and F2 shared some antigenic determinants, these two antisera were primarily directed against determinants unique to each protein. As shown in Fig. 2, anti-Fl reacted strongly with Fl but not detectably with F2, whereas, antiF2 reacted strongly with F2 and weakly with Fl. The relationship between Fl and F2 was more clearly indicated by two observations: (1) When released by cells in the absence of the protease inhibitor Trasylol, F1 purified under nondissociating conditions often migrated as several components in sodium dodecyl sulfate-gel electrophoresis. Such Fl molecules also dissociated into several pieces upon standing in physiological medium; the major component had the same electrophoretic mobility, molecular weight, and antigenic determinants as F2. (2) When intact F1 was partially degraded by trypsin (25 ,g/ml, 30 min, 370), a num-
Proc. Nat. Acad. Sci. USA 73 (1976)
Cell Biology: Rutishauser et al.
Anti - F 1
F1
F2 Anti - F 2
FIG. 2. Immunodiffusion analysis of F1 and F2 using antibodies to the purified proteins.
ber of fragments were produced, including F2. These results, along with the immunological and molecular weight data, indicate that monomeric F1 contains a protease-sensitive region that, when cleaved under nondenaturing conditions yields F2. Anti-Fl and anti-F2, although produced against purified proteins, reacted with a number of minor components in MCS and TCS. The proteins from MCS reacted more strongly with anti-Fl, and those from TCS reacted more strongly with anti-F2. All of these proteins shared antigenic determinants with F1 and/or F2. The data strongly suggest that these molecules are derived from the same molecule, and also confirm the specificity of the antisera. Identification and Characterization of F1 and F2 on the Cell Surface. Antibodies to F1 and F2 were used to study these and structurally related proteins on the surface of cells grown in suspension, monolayers, or intact tissue. In all three cases, fluorescein-labeled anti-Fl and anti-F2 stained retinal cells brain cells. These antibodies also stained chick embryo fibroblasts. They did not, however, stain chick embryo thymocytes or erythrocytes. Staining with anti-F2 was stronger than with anti-Fl, and on nearly all cells the fluorescence was distributed in small patches over the entire membrane. For correlation with cell-cell and cell-fiber binding experiments (see below), the amount of anti-Fl or anti-F2 bound by retinal and brain cells at particular stages of development was estimated in suspension using 125I-labeled antibody. Under saturating conditions (1 mg/ml of antibody), retinal cells from 8- and 13-day embryos and brain cells
from 6- and 10-day-old embryos bound on the average 2 to 4 X 105 anti-F2 molecules and 0.6 to 1.0 X 105 anti-Fl molecules per cell. As expected from these data on intact cells, anti-Fl and anti-F2 also precipitated 125I-labeled cell surface proteins extracted from retina cells (Fig. 3). When cells from monolayers were used, similar amounts of protein were precipitated by either antibody. With both antibodies this included a major component with a molecular weight in sodium dodecyl sulfate of 240,000, and minor components with molecular weights of 265,000, 175,000, and 65,000. When nondissociated retinal tissue or cells from suspension culture were extracted, however, most of the material precipitated by the same antibodies had a molecular weight in sodium dodecyl sulfate of 150,000, with a minor component of Mr 175,000; in this experiment, the anti-F2 precipitated about four times more protein than anti-Fl. Immunoglobulin from unimmunized rabbits did not precipitate significant amounts of protein. The data suggest that F1 and F2 are soluble cleavage products of a cell surface polypeptide, with a molecular weight of 240,000, and that in tissue or in suspension cultures the majority of these molecules are converted to polypeptides with a molecular weight of 150,000. In addition, the specific precipitation of components with molecular weights of 265,000 and 175,000 suggests that another cleavage, removing a fragment of Mr 25,000 from the membrane-bound molecules, may also have occurred. Binding among Retinal and Brain Cells from Embryos of Different Ages. In Table 1 are summarized the binding frequencies observed among trypsin-dissociated neural cells in the cell-cell binding assay and the effect of anti-F2 on this binding. High levels of binding were obtained between pairs of 8-day retinal cells, pairs of 6-day brain cells, and between pairs consisting of a 8-day retinal cell and a 6-day brain cell. If, however, the trypsinized cells were not allowed to recover in suspension cultures, no cell-cell binding occurred. Full recovery was achieved in 6-12 hr. In contrast, even fully recovered 13-day retinal cells and 10-day brain cells bound poorly to themselves and to each other. If 8-day retinal cells and 6-day brain cells were incubated with anti-F2 (1 mg/ml, 15 min, 250) prior to the assay, binding was inhibited by 75-90%. In contrast, preincubation with anti-Fl did not affect binding with either cell type. Binding of Cells to Fibers Coated with Anti-F2. The
Molecular weight (x 10-3) 50
Table 1. Binding among retinal and brain cells from embryos of different ages
250 200 50 100
1000I
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a
Cell-celli binding between: * Cell A Cell B
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Binding in
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anti-F2t
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02
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Relative mobility
FIG. 3. Sodium dodecyl sulfate-gel electrophoresis of 125I-labeled retinal cell surface proteins precipitated by anti-F2. The same proteins were precipitated by anti-Fl. (a) Proteins from trypsin-dissociated cells grown in monolayers. (b) Proteins from cells in undissociated retinal tissue. Molecular weights in sodium dodecyl sulfate-2-mercaptoethanol were assigned relative to the migration of 1311-labeled spectrin, f3-galactosidase, and immunoglobulin heavy chains in the same 6.5% polyacrylamide gels.
R8 *
Rs B6
B6
RI3 B10 RI3
B10
423 413 390 122 8 175 41
37 32 41
R8 and R13 are cells from retinas of 8- and 13-day-old chick embryos; B6 and B1o are cells from brains of 6- and 10-day-old em-
bryos.
t Expressed as number of B cells bound to 1 mm2 of A cell monolayer.
t Both Cell A and Cell B were incubated for 15 min at 250 with antibodies to F2 (1 mg/ml) prior to cell-cell binding.
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Proc. Nat. Acad. Sci. USA 73 (1976)
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Cleavage at B
w
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Fetal days
FIG. 4. Age dependence of cell binding to fibers coated with anti-F2. (a) Binding of retinal and brain cells to fibers as a function of embryonic age. The vertical axis (FBC/cm) indicates the number of cells bound along one edge of a one cm fiber segment. (b) Increase in the number of cells obtained by dissociation of retinal or brain tissue as a function of embryonic age.
ability of cells to bind to nylon fibers coated with anti-F2 also depended upon the age of the embryo, and different profiles were obtained for retinal and brain cells (Fig. 4a). Cells from both tissues of a 4-day embryo bound poorly to the fibers. The binding of brain cells increased to a maximum by 6 4dys and then decreased. Retinal cells did not bind until 5-6 days, reached maximal binding at 8-9 days, and then decreased in their binding to low levels by the 13th day. Binding of brain and retinal cells was inhibited 80-90% by preincubation of the anti-F2 fiber with F2 (20.ug/ml). In contrast to these results, binding of brain and retinal cells to fibers coated with the lectin concanavalin A or antibodies prepared against chick liver mlls that also bind to neural cells did not vary more than about 30% as a function of embryonic age. As with cell-cell binding, cells that were not allowed to recover from trypsinization did not bind to anti-F2 fibers. Maximum levels of binding were obtained after a recovery period of only 2-4 hr, in contrast to the cell-cell binding, which required 6-12 hr. In Fig. 4 is also shown a graph of the numbers of cells in a retina or brain as a function of embryonic age. Although these numbers may reflect a complex pattern of cell division, death, and migration, it is striking that, for both tissues, the rate of increase in cell number was temporally related to the ability of their cells to bind to other cells or to anti-F2 fibers. DISCUSSION
These studies of F1 and F2 and related proteins on brain and retinal cell surfaces can be interpreted in terms of the model shown in Fig. 5. This discussion, therefore, emphasizes the bases and the consequences of this model. The data on initial binding events among individual brain and retinal cells suggest that the molecular mechanism of cell adhesion is the same in these two tissues. Brain and retinal cells can bind to each other as well as to themselves, and in all cases this binding can be inhibited by anti-F2. The different temporal patterns displayed in the cell-cell binding
Cell-cell binding FIG. 5. Proposed schematic model for the formation of cellcell bonds by a cell adhesion molecule (CAM). Both the activation of CAM from proCAM and the production of soluble fragments F1, F2, and F3 from proCAM are shown. The F3 component has not been isolated and may be further degraded. Of the three possible mechanisms for CAM-mediated eell-cell binding, only the symmetrical CAM-CAM bond is shown. A description of these mechanisms as well as the assignment of cleavages and molecular weights is contained in the text. The dashed arrow indicates the blockage of cell-cell binding by reaction of anti-F2 with CAM. appear to be under control of factors other than simply the display of aggregation-promoting molecules unique to each tissue (17). In view of the specific inhibition of cell-cell binding by antibodies to F2, we suggest that structures on a cell surface molecule possessing antigenic determinants in common with F2 participate in the binding rNotion. This conclusion is further supported by the temporal correlation between the ability of a cell to bind to a fiber coated with anti-F2 and its ability to bind to another cell. The problem is therefore to relate the antigenic determinants on F2 to a cell surface molecule. In the model it is proposed that these determinants are present on a cell adhesion molecule (CAM), which contains F2 as an integral portion of its single polypeptide chain. CAM, which is found on cells in tissue or cultured in suspension, is generated by proteolytic cleavage of a precursor molecule, proCAM, which is found on the surface of retinal cells grown in monolayers. The model, therefore, depicts proCAM as having at least two regions that are sensitive to proteolytic cleavage. One of these cleavage sites, called B in Fig. 5, involves the release of fragment F1 into culture medium. The other, called the A site, would account for two cleavages; (1) The formation of the F2 subunit from F1, and (2) The conversion of proCAM into CAM. In solution, the A site cleavage releases the F2 subunit that subsequently forms a dimer. The same cleavage of proCAM would produce the cell surface molecule CAM, which
experiments, therefore,
Cell Biology: Rutishauser et al. carries the antigenic determinants involved in cell-cell binding.
The ability of anti-F2 but not anti-Fl to inhibit cell-cell binding is consistent with the proposed model. Anti-F2 reacts most strongly with the F2-related determinants on CAM and, therefore, prevents CAM-mediated cell-cell binding. On the other hand, anti-Fl antibodies do not react with F2 antigenic determinants, and would not necessarily be expected to block adhesion. Assuming that CAM participates directly in cell adhesion, it still remains to be specified how cell-cell bonds are formed. There are three main possibilities: (1) CAMs on different cells bind directly to each other. (2) CAMs on different cells are bridged by an as yet unidentified molecule. (3) CAMs on one cell bind to an unidentified receptor on another cell. At this time, we favor the first mechanism because of its simplicity and because F2 exists as a dimer in solution. It should be noted, however, that it has not been proven that binding can occur between two CAMs. The activation of proCAM by proteolysis raises the possibility that cell adhesion is controlled by mechanisms similar to those observed in blood clotting and complement fixation. The presence of many Fl- and F2-related fragments in MCS and TCS suggests that the cleavage mechanisms could be more complex than shown in Fig. 5. Furthermore, breakage of cell-cell adhesions might involve cleavage at site B, with the consequent release of F2 dimers. The correlation between the binding of cells to fibers coated with anti-F2 and their ability to bind to other cells suggests that another mechanism may also control cell adhesion. The amount of soluble anti-F2 and anti-Fl that binds to the surface of cells from brain and retina did not change significantly with embryonic development. At first glance, this observation would seem to be inconsistent with the temporal variation observed in the cell-cell and cell-fiber assays. It has been shown, however, that the distribution or mobility of molecules at the cell surface can affect their ability to form cell-cell and cell-fiber bonds (13, 18). In this respect it is noteworthy that the mobility of cell surface molecules can be modulated by a network of cytoplasmic microtubules and microfilaments (19). While it has not been established whether such a network is involved in morphogenesis, it is clear that receptor properties such as mobility or distribution must be considered in analyzing cell adhesion. It is particularly striking that the temporal increase in cell numbers in brain or retinal tissues was correlated with increases in cell-cell and cell-fiber binding. This suggests that changes in cell surface properties coordinated with the cell cycle may also affect cell adhesion. In view of its molecular weight and presence on the cell surface, proCAM may resemble the transformation-sensitive protein described by a number of laboratories (see ref. 20). In fact, it has been suggested that this protein, which has a molecular weight of 250,000 and spans the cell membrane (21), might in some way be involved with modulation of re-
Proc. Nat. Acad. Sci. USA 73 (1976)
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ceptor mobility, growth control, and agglutination reactions (22). The two molecules appear to differ significantly in the size of their cleavage products, however, and we have not been able to detect any immunological crossreaction between them. Nevertheless, the information available so far is not sufficient to rule out the possibility that they are related. In any event, it will be important to determine whether proCAM spans the cell membrane and is associated with intracellular networks of microtubules and microfilaments comprising a cell surface modulating assembly (23). We are grateful to Ms. Mildred Decker and Ms. Helvi Hjelt for excellent technical assistance. This work was supported by U.S. Public Health Service Grants HD 09635, Al 11378, Al 09273, AM 04256. J.-P.T. is a fellow of the International Agency for Research on Cancer, Lyon, France, R.B. is a fellow of the Jane Coffin Childs Memorial Fund for Medical Research, and B.S. is supported by a fellowship from the Damon Runyon-Walter Winchell Cancer Fund. 1. Weiss, L. (1968) The Cell Periphery, Metastasis and Other Contact Phenomena, eds. Neuberger, A. & Tatum, E. L. (American Elsevier, New York). 2. Moscona, A. A. (1973) in Cell Biology in Medicine, ed. Bittar, E. E. (Wiley-Interscience, New York), pp. 571-591. 3. Steinberg, M. S. (1970) J. Exp. Zool. 173,395-434. 4. Curtis, A. S. G. (1967) The Cell Surface (Academic Press, New York). 5. Balsamo, J. & Lilien, J. (1974) Nature 251, 522-524. 6. Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-436. 7. Stephens, R. E. (1975) Anal. Biochem. 65,369-379. 8. Cebra, J. J. & Goldstein, G. (1965) J. Immunol. 95, 230-245. 9. McConahey, P. J. & Dixon, F. J. (1966) Int. Arch. Allergy Appl. Immunol. 29, 185-189. 10. Henning, R., Milner, R. J., Reske, K., Cunningham, B. A. & Edelman, G. M. (1976) Proc. Nat. Acad. Sci. USA 73, 118122. 11. Laemmli, U.K. (1970) Nature 227,680-685. 12. Edelman, G. M. & Rutishauser, U. (1974) in Methods in Enzymology, eds. Jakoby, W. & Wilchek, M. (Academic Press, New York), pp. 195-225. 13. Rutishauser, U. & Sachs, L. (1974) Proc. Nat. Acad. Sci. USA
71,2456-2460. 14. Sela, B., Lis, H., Sharon, N. & Sachs, L. (1973) Biochim. Biophys. Acta 310, 273-277. 15. Bodmer, W. F., Tripp, M. & Bodmer, J. (1967) in Histocompatibility Testing, 1967 (Munksgaard, Copenhagen), pp. 341-350. 16. Ackers, G. A. (1970) Adv. Protein Chem. 24,343-446. 17. Rutishauser, U., Yang, D., Thiery, J.-P. & Edelman, G. M. (1975) in Proceedings of the 1975 Conference on Development Biology, ICN-UCLA Symposia, in press. 18. Rutishauser, U. & Sachs, L. (1975) J. Cell Biol. 66,76-85. 19. Edelman, G. M., Yahara, I. & Wang, J. L. (1973) Proc. Nat. Acad. Sci. USA 70,1442-1446. 20. Hynes, R. 0. (1974) Cell 1, 147-155. 21. Hunt, R. C. & Brown, J. C. (1975) J. Mol. Biol. 97, 413-422. 22. Yamada, K. M., Yamada, S. S. & Pastan, I. (1975) Proc. Nat. Acad. Sci. USA 72,3158-3162. 23. Edelman, G. M., Wang, J. L. & Yahara, I. (1975) "Cell motility," Cold Spring Harbor Symp. Quant. Biol., in press.
