DEVELOPMENTAL
BIOLOGY
T&206-216
(1979)
Immunological Detection of Cell Surface Components Related with Aggregation of Chinese Hamster and Chick Embryonic Cells HIDEKO URUSHIHARA, HIROKI S. OZAKI,’ AND MASATOSHI TAKEICHI Department
of Biophysics,
Faculty
of Science, University
of Kyoto, Kyoto 606, Japan
Received August 2, 1978; accepted in revised form December 14, 1978 Previous studies suggested that Chinese hamster V79 cells possess two mechanisms for their mutual adhesion, Ca’+-dependent and Ca2+-independent ones. We could prepare cells with only the Ca*‘-dependent mechanism intact by dispersing cell monolayers with trypsin (0.01%) containing Ca*+. In the present study, we found that cells dispersed with a very low concentration of trypsin (0.0001%) in the absence of Ca2+ retain only the Ca’+-independent mechanism intact. Fab fragments of antibodies directed against surface antigens of V79 cells inhibited the aggregation of V79 cells by the Ca*+-independent mechanism, but did not inhibit the aggregation of these cells by the Ca’+-dependent mechanism. These results suggest that the two mechanisms of cell adhesion are based on different cellular components. Molecules responsible for the Cap+-independent adhesion mechanism are probably cell surface components, because they were released from cells by the treatment with 0.01% trypsin without losing their specific antigenicity. The presence of adhesion mechanisms similar to those in V79 cells was shown in neural retinal cells of chick embryos. It was assumed, therefore, that these mechanisms of cell adhesion are generally present among a variety of cell types. INTRODUCTION
Adhesiveness between cells is an essential property of multicellular animal cells for the architecture of tissues and organs as well as for establishing interaction systems between cells (Trinkaus, 1969). Studies on the mechanisms of cell adhesion, therefore, are basically related to an understanding of various developmental events. The use of cell lines as a model system is of great advantage for this purpose, because a large homogeneous cell population is always available when necessary, and use of these cells guarantees well the reproducibility of results. In experiments using Chinese hamster V79 cells (Takeichi, 1977) and BHK cells (Urushihara et al., 1977; Ozaki et al., 1978), we recently suggested that a single cell is provided with at least two different mechanisms, Ca2’-dependent and Ca”-independent ones, for their mutual adhesion. ’ Present address: Department of Anatomy, Faculty of Medicine, University of Kyoto, Kyoto 606, Japan. 206 OOE-1606/79/050206-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reuroduction in any form reserved.
Both mechanisms are trypsin-sensitive, but the Ca2+-dependent one becomes trypsinresistant in the presence of Ca2’. Accordingly, cells dispersed with trypsin (0.01%) in the presence of Ca”’ retained only the Ca2’dependent mechanism, whereas cells dispersed with trypsin (0.01%) in the absence of Ca2+ lost both mechanisms. Cells dispersed with EDTA seemed to retain both mechanisms. In the present report, we precisely reexamined the trypsin sensitivity of these adhesion mechanisms in V79 cells, and found a special condition for cell dispersion in which only the Ca2+-independent mechanism remains intact in cells. It is thus possible to obtain cells with either one of the two adhesion mechanisms. By comparing those cells, we investigated the possibility that there are specific cell surface molecules that function for each mechanism. The immunological method, which has been employed for analyses of cell adhesion mechanisms by several workers (Spiegel, 1954a, b; Beug et al., 1973; McClay et al.,
URUSHIHARA,
OZAKI,
AND
TAKEICHI
1977; Brackenbury et al., 1977; Thiery et al., 1977), was adopted for this purpose. In addition, we studied whether or not the assumption of the presence of two separable mechanisms of cell adhesion is applicable to freshly dissociated embryonic neural retinal cells.
Cell Adhesion
Mechanisms
207
ously shown that E-, TC-, and TE-V79 are different in aggregative properties; E-V79 aggregate both in the presence and absence of Ca2+ in the aggregating media, TC-V79 aggregate only in the presence of Ca’+, and TE-V79 do not aggregate either in the presence or in the absence of Ca”+ (Takeichi, 1977; also see Table 1). MATERIALS AND METHODS Dispersion of neural retinal cells. Neural retinas (NR) from 7-day-old chick embryos Culture and dispersion of V79 cells. V79 were treated in four different ways with the cells, a Chinese hamster lung cell line (Yu, following dissociation media: (1) 1 m&f 1963), were grown on glass petri dishes in EDTA in HCMF, (2) 0.01% crystalline trypEagle’s MEM (2 x amino acids and vitamins) supplemented with 6% fetal calf se- sin plus 10 mM CaC12 in HCMF, (3) 0.01% crystalline trypsin plus 1 mM EDTA in rum (Grand Island Biological Co.). HCMF and (4) 0.0005% crystalline trypsin Cell monolayers were dispersed into sinplus 1 n&f EDTA in HCMF. Cells treated gle cell suspensions with the following four with these media are designated as E-NR, different treatments, the resulting cells TC-NR, TE-NR, or LTE-NR, respectively. being designated as E-, TC-, TE-, or LTEPrior to the above treatment, isolated V79 (Takeichi, 1977). (1) In the EDTA tissues were rinsed with HCMF suppletreatment, a cell monolayer was rinsed three times with Ca”+- and Mg2+-free mented with the reagents present in the Puck’s saline (CMF) and incubated with 1 dissociation media, either 1 m&f EDTA or 10 mJ4 CaC12. Of the dissociation medium, mJ4 EDTA in Ca2+- and Mg”‘-free saline 0.5 ml was added to pieces of neural retina buffered with Hepes (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) at pH obtained from one eye. The incubation was 7.4 (HCMF). (2) For the trypsin-Ca”’ treatcarried out in a reciprocal shaker (140 ment, a cell monolayer was rinsed three strokes/min) for 60 min at 37°C with a times with CMF containing 1 mM CaClz gentle pipetting of tissues every 20 min. The dispersed cells were collected and and incubated with a solution containing both 0.01% crystalline trypsin (type I, washed as in V79 cells, and suspended in Sigma Chemical Co.) and 0.1 or 1 mM CaC12 HCMF. Large clumps which remained in HCMF. (3) In the trypsin-EDTA treatwithout being dispersed were removed by ment, a cell monolayer rinsed three times filtration through four layers of gauze. Cell aggregation. Plastic wells (Disposo with CMF was incubated with 0.01% crystray Model FB16-24TC, Linbro Scientific talline trypsin plus 1 m&f EDTA in HCMF. Co., Inc.), which had previously been (4) For the light trypsin-EDTA treatment, 0.0001% trypsin was used in place of the coated with bovine serum albumin to pre0.01% trypsin used in method (3). In all vent attachment of cells to the surface these treatments cells were incubated at (Takeichi and Okada, 1972), were used for 37°C for 15 min on a gyratory shaker at 80 the aggregation assay. Into each well 4 x lo” V79 cells or 3 X 10” NR cells suspended rpm. Cells detached from culture plates were collected, centrifuged, and resus- in 0.5 ml of HCMF were inoculated and pended in CMF which contained 0.05% or incubated at 37°C on a gyratory shaker at 0.0005% soybean trypsin inhibitor (type I- 80 rpm. Other reagents, when necessary, S, Sigma Chemical Co.) for preparing TEwere added to the aggregation medium just and TC-V79 or LTE-V79. Cells were before the start of incubation usually from washed two more times with CMF and loo-fold concentrated stock solutions. After the indicated &me of incubation, the total finally suspended in HCMF. It was previ-
208
DEVELOPMENTAL BIOLOGY
particle number in a given cell suspension of each well was counted with a Coulter counter (Model Zg, Coulter Electronics Inc.) with a 100~pm aperture. The degree of aggregation was represented by the ratio of the total particle number at incubation time t (Nt) to the initial single cell number (No). Data were represented by the mean of values obtained from four identical samples. For details of the method for the aggregation assay, see previous papers (Ueda and Takeichi, 1976; Urushihara et al., 1976, 1977; Takeichi, 1977). Immunization procedure and preparation of antibodies. Rabbits were immunized at weekly intervals with 1 x lo8 E-V79 or Ehlrich ascites tumor cells (EAT) by the following schedule: (1) intravenous injection of freshly dispersed cells suspended in 0.8 ml of HCMF supplemented with 1 mM CaCL; (2) intramuscular injection of the mixture of cells fixed with 2% glutaraldehyde in HCMF and Freund’s complete adjuvant; (3) intravenous and subcutaneous injection of fresh cells; (4) intramuscular injection of fixed cells in Freund’s incomplete adjuvant; and (5) the same as step (3). One week after the fifth injection and at monthly intervals thereafter, antiserum was collected and stored frozen. Activity of anti-E-V79 was assayed by titrating its ability to agglutinate E-V79 after heat inactivation. The degree of cell clumping in antiserum much exceeded that solely due to the spontaneous aggregation of cells, so that it was possible to detect the cell agglutinating activity of antiserum. Activity of anti-EAT was assayed by agglutination of EAT. This antiserum was found also to agglutinate E-V79 to the same degree. Antisera with maximum cell agglutinating activity at a 10e3 dilution was used for the experiments. The immunoglobulin G (IgG) fraction of antisera was obtained by ammonium sulfate precipitation followed by DEAE-cellulose column chromatography according to Fahey (1967). Fab fragments of IgG were prepared according to the procedure of Ut-
VOLUME 70,1979
sumi (1969). Ten milligrams per milliliter of IgG was incubated with 0.1 mg/ml papain (Worthington) in 50 mM sodium acetate buffer, pH 4.5, containing 28 n&f 2mercaptoethanol at 37°C for 24 hr. After being adjusted to pH 8.0, the digest was fractionated with Sephadex G-100. Fab fraction was collected, dialyzed against 10 m&f NH4HC03, and lyophilized. Throughout this work antibodies were used as Fab fragments. Absorption of antibodies by dispersed cells. Suspension of 5 x lo7 V79 cells dispersed by different treatments were pelleted by centrifugation at 500g for 5 min, and the supernatant was replaced with 0.8 ml of 1.25 mg/ml Fab solution in HCMF containing 20 U/ml Trasylol (Calbiochem). After dispersing the cell pellet by flushing with a pipet, the Fab-cell mixture was incubated at 0°C for 2 hr with occasional shaking. At the end of incubation cells were removed by centrifugation at 500g for 5 min. The supernatant was further centrifuged for clarification at 15,000g for 15 min, dialyzed against HCMF, and used as absorbed Fab. Preparation of cell trypsinates. In order to collect trypsin-sensitive materials on cell surfaces, dispersed V79 cells were further treated with trypsin. In 2 ml of HCMF containing 0.01% trypsin and 1 n-J4 EDTA or 0.1 mM CaC12, 1 X 10’ cells were suspended and incubated in a reciprocating shaker (140 strokes/min) at 37°C for 15 min. After centrifugation at 500g for 5 min, the supernatant was recentrifuged for clarification at 15,OOOg for 15 min and was applied to a trypsin inhibitor column (see below). Prior to the application to the column, 1 M NaCl was added to the sample to prevent nonspecific absorption of proteins. Unbound material, which will be called cell trypsinate, was collected, dialyzed against 10 mJ4 NH4HC03, and lyophilized. In cases where the second treatment was done with trypsin plus Ca”, cells were incubated in 1 mM CaClz for 10 min prior to this treatment.
URUSHIHARA,
OZAKI,
AND
TAKEICHI
Preparation of trypsin-inhibitor affinity column. In order to prepare an affinity column of trypsin inhibitor, 10 mg of soybean trypsin inhibitor was coupled with 1 g of CNBr-activated Sepharose 4B (Pharmacia) following the method recommended by the company. One milliliter of coupled gel was packed in a 2-ml plastic syringe. The capacity of this trypsin-inhibitor column was large enough to be used for the present purpose, as checked by assaying the activity of trypsin bound to it by a spectrophotometric method (Schwert and Takenaka, 1955). RESULTS
Preparation of V79 Cells with only Ca2+independent Adhesion Mechanism In order to determine the critical concentration of trypsin to inactivate the Ca’+independent adhesion mechanism, cell monolayers were treated with various concentrations of trypsin in the presence of either 1 m&f EDTA or 1 mJ4 Ca2+ at 37°C for 15 min. The aggregating ability of these cells was assayed in the Ca2+-free medium (HCMF) to selectively detect the Ca2+-independent process of cell adhesion. As shown in Fig. 1, curve A, trypsin at concentrations higher than 0.01% was required to make cells completely nonadhesive in Ca*‘free conditions. Whether EDTA or Ca2+ was added to the trypsin solution did not affect the trypsin sensitivity of the Ca2’independent adhesion property of cells, as suggested previously (Takeichi, 1977). The trypsin sensitivity of the Ca2+-dependent mechanism cannot be assayed in the same system as above, because the Ca2+-independent mechanism which also operates in the aggregation of cells interferes with the detection of the Ca’+-dependent adhesion process. Therefore, cells having only the Ca2+-dependent adhesion property (TC-V79) were prepared first by the trypsin-Ca2+ treatment of Takeichi (1977). Then these cells were retreated with various concentrations of trypsin in the presence of EDTA or EGTA, to determine the
Cell Adhesion
Mechanisms
209
Trypsin mm. (‘1. I
FIG. 1. Trypsin sensitivity of the adhesive properties of V79 cells. (A) Aggregation of cells dispersed with trypsin at the concentrations indicated on the abscissa in the presence of 1 mIi4 EDTA. (B) Aggregation of cells which were once dispersed with the trypsin-Ca*+ treatment and received a retreatment with trypsin (at concentrations indicated on the abscissa) plus 1 m&f EDTA. The method of retreatment with trypsin was the same as that described in footnote a to Table 2. Both in (A) and (B), 1 x 10’ cells were treated with 5 ml of trypsin solution. Aggregation was assayed after 2 hr of gyration in Ca’+-free aggregation medium (A) or in Ca*’ (1 n&f)-containing medium (B).
critical concentration of trypsin for inactivation of the Ca2+-dependent adhesion mechanism. As shown by curve B in Fig. 1, the Ca”-dependent adhesive property of cells is very sensitive to trypsin; 0.0001% of trypsin was enough to remove this adhesion property from cells. If Ca2+ (1 mJ4) was added to the trypsin solution, Ca2+-dependent adhesiveness was not inactivated at all at any concentration of trypsin tested. As EDTA had the same effects as EGTA, the former chelater was used in the following experiments described-in this report. To summarize, the critical concentration of trypsin needed to abolish the Ca2+-independent adhesion property is O.Ol%, and that needed to abolish the Ca2+-dependent adhesion property is 0.0001% in Ca2+-free medium. On the basis of these results, it can be predicted that cells dispersed with 0.0001% trypsin containing 1 mM EDTA (light trypsin-EDTA treatment) directly from cell monolayers do not have the Ca’+dependent mechanism of adhesion but retain the Ca2’-independent one. Figure 2 shows the aggregation kinetics of V79 cells
210
DEVELOPMENTALBIOLOGY
dispersed with this light trypsin-EDTA treatment (LTE-V79). As expected, aggregation of these cells was completely Ca’+independent. These cells aggregated also at a low temperature (4’33, which is characteristic of aggregation by the Ca2+-independent mechanism (Takeichi, 1977). In Table 1, the aggregation properties of V79 cells dispersed with four different treatments, three of which had already been published (Takeichi, 1977), were summarized. We postulated from previous work (Takeichi, 1977) that E-V79 have both the Ca2+-dependent and Ca”-independent mechanisms, TC-V79 have only the Ca*+dependent mechanism, and TE-V79 have neither one. LTE-V79 are assumed to have only the Ca2’-independent mechanism from the above results. To reconfirm these
VOLUME 70,1979
assumptions, the following experiments were performed. If our assumptions are correct, cells with only the Ca2+-dependent mechanism of adhesion or cells with only the Ca2’-independent mechanism should be obtained from E-V79 by the appropriate treatment with trypsin. As shown in Table 2, E-V79 retreated with “trypsin-Ca”” aggregated TABLE 2 EFFECTOF TRYPSIN TREATMENTSON ADHESIVE PROPERTYOF ONCE-DISPERSEDV79 CELLS Cells Second treatment” Aggregation treated WdNo) *
E-V79
TC-V79 LTE-V79
-Co’+ I 0
1 30 Time
I
I
60
120 lmln)
FIG. 2. Aggregation of V79 cells dispersed with O.OOOl% trypsin + 1 mMEDTA (LTE-V79) in medium either with 1 mM Ca’+ (0) or without Ca*+ (0).
Trypsin-Ca*’ Light trypsin-EDTA EDTA Light trypsin-EDTA Trypsin-Ca2+ Trypsin-Ca2+ Light trypsin-EDTA
With Ca2+
With. out t-h=+ --
0.325 0.301 0.211 0.890 0.186 0.995 0.285
0.998 0.307 0.350 0.837 l.OOO 0.992 0.273
“Cells once dispersed by appropriate treatments were collected in a centrifuge tube and incubated with the reagents for the second treatment at 37°C for 15 min. In the case of trypsin-Ca*+ treatment, cells were incubated in 1 mM Ca” for 10 min prior to the second treatment. Retreated cells were washed in the same way as the first treatment and finally suspended in HCMF. Trypsin-Ca2+ = 0.01% trypsin + 1 mM Ca*+; light trypsin-EDTA = O.OOOl%trypsin + 1 mM EDTA; EDTA = 1 mM EDTA. Cells which received a second treatment with trypsin-EDTA (0.01%trypsin + 1 mM EDTA) lost their adhesiveness completely in every case. b Shown by aggregation index after a 2-hr incubation.
TABLE 1 ADHESIVE PROPERTIESOF V79 CELLS DISPERSEDWITH DIFFERENT TREATMENTS Abbreviated cell Presumed adhesion mechanisms Dispersion treatment” Aggregation* intact” name With Ca*+ Wg-rgut E-V79 TC-V79 LTE-V79 TE-V79
EDTA Trypsin-Ca*+ Light trypsin-EDTA Trypsin-EDTA
+++ +++ +++ -
-i--l+++
Ca”-dep and Ca”-indep Ca’+-dep Ca”-indep None
a EDTA = 1 mM EDTA; Trypsin-Ca2+ = 0.01%trypsin + 1 m&f Ca’+; light trypsin-EDTA + 1 mM EDTA; trypsin-EDTA = 0.01% trypsin + 1 mlM EDTA. ’ Data for E-, TC-, and TE-V79 are from Takeichi (1977). ’ Ca”-dep = Ca”-dependent mechanism; Ca2+-mdep = Ca*+-independent mechanism.
= O.OOOl% trypsin
URUSHIHARA,
OZAKI,
AND
Cell Adhesion
TAKEICHI
centration of Fab, although less than in the control, as shown in Fig. 3a. The Fab strongly inhibited the aggregation of LTEV79 both in the presence and absence of Ca2+. The aggregation of TC-V79, however, was not affected at all (Figs. 3c and 3b). The inhibitory effect of Fab was dependent on its concentration as shown in Fig. 4, the
only in the presence of Ca”, while E-V79 retreated with “light trypsin-EDTA” aggregated to the same degree in the presence and absence of Ca2+. As is also shown in Table 2, TC-V79 retreated with “light trypsin-EDTA” and LTE-V79 retreated with “trypsin-Ca’+” lost their adhesiveness completely. These results are totally consistent with the above assumptions on the distribution of two adhesion mechanisms in cells dispersed with different treatments as shown in Table 1. The second trypsin treatment in these experiments did not increase the dead cell population significantly, as judged from growing ability of the cells which received the second treatment in normal culture medium. Usually, more than 90% of the cells were alive at the end of the above experiments.
0
0 001
0 01 Fab
Inhibition of Vi’9 Cell Aggregation by Fab Fragments of Anti-E-V79 Antibody
01 con
BIOLOGY
T&206-216
(1979)
Immunological Detection of Cell Surface Components Related with Aggregation of Chinese Hamster and Chick Embryonic Cells HIDEKO URUSHIHARA, HIROKI S. OZAKI,’ AND MASATOSHI TAKEICHI Department
of Biophysics,
Faculty
of Science, University
of Kyoto, Kyoto 606, Japan
Received August 2, 1978; accepted in revised form December 14, 1978 Previous studies suggested that Chinese hamster V79 cells possess two mechanisms for their mutual adhesion, Ca’+-dependent and Ca2+-independent ones. We could prepare cells with only the Ca*‘-dependent mechanism intact by dispersing cell monolayers with trypsin (0.01%) containing Ca*+. In the present study, we found that cells dispersed with a very low concentration of trypsin (0.0001%) in the absence of Ca2+ retain only the Ca’+-independent mechanism intact. Fab fragments of antibodies directed against surface antigens of V79 cells inhibited the aggregation of V79 cells by the Ca*+-independent mechanism, but did not inhibit the aggregation of these cells by the Ca’+-dependent mechanism. These results suggest that the two mechanisms of cell adhesion are based on different cellular components. Molecules responsible for the Cap+-independent adhesion mechanism are probably cell surface components, because they were released from cells by the treatment with 0.01% trypsin without losing their specific antigenicity. The presence of adhesion mechanisms similar to those in V79 cells was shown in neural retinal cells of chick embryos. It was assumed, therefore, that these mechanisms of cell adhesion are generally present among a variety of cell types. INTRODUCTION
Adhesiveness between cells is an essential property of multicellular animal cells for the architecture of tissues and organs as well as for establishing interaction systems between cells (Trinkaus, 1969). Studies on the mechanisms of cell adhesion, therefore, are basically related to an understanding of various developmental events. The use of cell lines as a model system is of great advantage for this purpose, because a large homogeneous cell population is always available when necessary, and use of these cells guarantees well the reproducibility of results. In experiments using Chinese hamster V79 cells (Takeichi, 1977) and BHK cells (Urushihara et al., 1977; Ozaki et al., 1978), we recently suggested that a single cell is provided with at least two different mechanisms, Ca2’-dependent and Ca”-independent ones, for their mutual adhesion. ’ Present address: Department of Anatomy, Faculty of Medicine, University of Kyoto, Kyoto 606, Japan. 206 OOE-1606/79/050206-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reuroduction in any form reserved.
Both mechanisms are trypsin-sensitive, but the Ca2+-dependent one becomes trypsinresistant in the presence of Ca2’. Accordingly, cells dispersed with trypsin (0.01%) in the presence of Ca”’ retained only the Ca2’dependent mechanism, whereas cells dispersed with trypsin (0.01%) in the absence of Ca2+ lost both mechanisms. Cells dispersed with EDTA seemed to retain both mechanisms. In the present report, we precisely reexamined the trypsin sensitivity of these adhesion mechanisms in V79 cells, and found a special condition for cell dispersion in which only the Ca2+-independent mechanism remains intact in cells. It is thus possible to obtain cells with either one of the two adhesion mechanisms. By comparing those cells, we investigated the possibility that there are specific cell surface molecules that function for each mechanism. The immunological method, which has been employed for analyses of cell adhesion mechanisms by several workers (Spiegel, 1954a, b; Beug et al., 1973; McClay et al.,
URUSHIHARA,
OZAKI,
AND
TAKEICHI
1977; Brackenbury et al., 1977; Thiery et al., 1977), was adopted for this purpose. In addition, we studied whether or not the assumption of the presence of two separable mechanisms of cell adhesion is applicable to freshly dissociated embryonic neural retinal cells.
Cell Adhesion
Mechanisms
207
ously shown that E-, TC-, and TE-V79 are different in aggregative properties; E-V79 aggregate both in the presence and absence of Ca2+ in the aggregating media, TC-V79 aggregate only in the presence of Ca’+, and TE-V79 do not aggregate either in the presence or in the absence of Ca”+ (Takeichi, 1977; also see Table 1). MATERIALS AND METHODS Dispersion of neural retinal cells. Neural retinas (NR) from 7-day-old chick embryos Culture and dispersion of V79 cells. V79 were treated in four different ways with the cells, a Chinese hamster lung cell line (Yu, following dissociation media: (1) 1 m&f 1963), were grown on glass petri dishes in EDTA in HCMF, (2) 0.01% crystalline trypEagle’s MEM (2 x amino acids and vitamins) supplemented with 6% fetal calf se- sin plus 10 mM CaC12 in HCMF, (3) 0.01% crystalline trypsin plus 1 mM EDTA in rum (Grand Island Biological Co.). HCMF and (4) 0.0005% crystalline trypsin Cell monolayers were dispersed into sinplus 1 n&f EDTA in HCMF. Cells treated gle cell suspensions with the following four with these media are designated as E-NR, different treatments, the resulting cells TC-NR, TE-NR, or LTE-NR, respectively. being designated as E-, TC-, TE-, or LTEPrior to the above treatment, isolated V79 (Takeichi, 1977). (1) In the EDTA tissues were rinsed with HCMF suppletreatment, a cell monolayer was rinsed three times with Ca”+- and Mg2+-free mented with the reagents present in the Puck’s saline (CMF) and incubated with 1 dissociation media, either 1 m&f EDTA or 10 mJ4 CaC12. Of the dissociation medium, mJ4 EDTA in Ca2+- and Mg”‘-free saline 0.5 ml was added to pieces of neural retina buffered with Hepes (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) at pH obtained from one eye. The incubation was 7.4 (HCMF). (2) For the trypsin-Ca”’ treatcarried out in a reciprocal shaker (140 ment, a cell monolayer was rinsed three strokes/min) for 60 min at 37°C with a times with CMF containing 1 mM CaClz gentle pipetting of tissues every 20 min. The dispersed cells were collected and and incubated with a solution containing both 0.01% crystalline trypsin (type I, washed as in V79 cells, and suspended in Sigma Chemical Co.) and 0.1 or 1 mM CaC12 HCMF. Large clumps which remained in HCMF. (3) In the trypsin-EDTA treatwithout being dispersed were removed by ment, a cell monolayer rinsed three times filtration through four layers of gauze. Cell aggregation. Plastic wells (Disposo with CMF was incubated with 0.01% crystray Model FB16-24TC, Linbro Scientific talline trypsin plus 1 m&f EDTA in HCMF. Co., Inc.), which had previously been (4) For the light trypsin-EDTA treatment, 0.0001% trypsin was used in place of the coated with bovine serum albumin to pre0.01% trypsin used in method (3). In all vent attachment of cells to the surface these treatments cells were incubated at (Takeichi and Okada, 1972), were used for 37°C for 15 min on a gyratory shaker at 80 the aggregation assay. Into each well 4 x lo” V79 cells or 3 X 10” NR cells suspended rpm. Cells detached from culture plates were collected, centrifuged, and resus- in 0.5 ml of HCMF were inoculated and pended in CMF which contained 0.05% or incubated at 37°C on a gyratory shaker at 0.0005% soybean trypsin inhibitor (type I- 80 rpm. Other reagents, when necessary, S, Sigma Chemical Co.) for preparing TEwere added to the aggregation medium just and TC-V79 or LTE-V79. Cells were before the start of incubation usually from washed two more times with CMF and loo-fold concentrated stock solutions. After the indicated &me of incubation, the total finally suspended in HCMF. It was previ-
208
DEVELOPMENTAL BIOLOGY
particle number in a given cell suspension of each well was counted with a Coulter counter (Model Zg, Coulter Electronics Inc.) with a 100~pm aperture. The degree of aggregation was represented by the ratio of the total particle number at incubation time t (Nt) to the initial single cell number (No). Data were represented by the mean of values obtained from four identical samples. For details of the method for the aggregation assay, see previous papers (Ueda and Takeichi, 1976; Urushihara et al., 1976, 1977; Takeichi, 1977). Immunization procedure and preparation of antibodies. Rabbits were immunized at weekly intervals with 1 x lo8 E-V79 or Ehlrich ascites tumor cells (EAT) by the following schedule: (1) intravenous injection of freshly dispersed cells suspended in 0.8 ml of HCMF supplemented with 1 mM CaCL; (2) intramuscular injection of the mixture of cells fixed with 2% glutaraldehyde in HCMF and Freund’s complete adjuvant; (3) intravenous and subcutaneous injection of fresh cells; (4) intramuscular injection of fixed cells in Freund’s incomplete adjuvant; and (5) the same as step (3). One week after the fifth injection and at monthly intervals thereafter, antiserum was collected and stored frozen. Activity of anti-E-V79 was assayed by titrating its ability to agglutinate E-V79 after heat inactivation. The degree of cell clumping in antiserum much exceeded that solely due to the spontaneous aggregation of cells, so that it was possible to detect the cell agglutinating activity of antiserum. Activity of anti-EAT was assayed by agglutination of EAT. This antiserum was found also to agglutinate E-V79 to the same degree. Antisera with maximum cell agglutinating activity at a 10e3 dilution was used for the experiments. The immunoglobulin G (IgG) fraction of antisera was obtained by ammonium sulfate precipitation followed by DEAE-cellulose column chromatography according to Fahey (1967). Fab fragments of IgG were prepared according to the procedure of Ut-
VOLUME 70,1979
sumi (1969). Ten milligrams per milliliter of IgG was incubated with 0.1 mg/ml papain (Worthington) in 50 mM sodium acetate buffer, pH 4.5, containing 28 n&f 2mercaptoethanol at 37°C for 24 hr. After being adjusted to pH 8.0, the digest was fractionated with Sephadex G-100. Fab fraction was collected, dialyzed against 10 m&f NH4HC03, and lyophilized. Throughout this work antibodies were used as Fab fragments. Absorption of antibodies by dispersed cells. Suspension of 5 x lo7 V79 cells dispersed by different treatments were pelleted by centrifugation at 500g for 5 min, and the supernatant was replaced with 0.8 ml of 1.25 mg/ml Fab solution in HCMF containing 20 U/ml Trasylol (Calbiochem). After dispersing the cell pellet by flushing with a pipet, the Fab-cell mixture was incubated at 0°C for 2 hr with occasional shaking. At the end of incubation cells were removed by centrifugation at 500g for 5 min. The supernatant was further centrifuged for clarification at 15,000g for 15 min, dialyzed against HCMF, and used as absorbed Fab. Preparation of cell trypsinates. In order to collect trypsin-sensitive materials on cell surfaces, dispersed V79 cells were further treated with trypsin. In 2 ml of HCMF containing 0.01% trypsin and 1 n-J4 EDTA or 0.1 mM CaC12, 1 X 10’ cells were suspended and incubated in a reciprocating shaker (140 strokes/min) at 37°C for 15 min. After centrifugation at 500g for 5 min, the supernatant was recentrifuged for clarification at 15,OOOg for 15 min and was applied to a trypsin inhibitor column (see below). Prior to the application to the column, 1 M NaCl was added to the sample to prevent nonspecific absorption of proteins. Unbound material, which will be called cell trypsinate, was collected, dialyzed against 10 mJ4 NH4HC03, and lyophilized. In cases where the second treatment was done with trypsin plus Ca”, cells were incubated in 1 mM CaClz for 10 min prior to this treatment.
URUSHIHARA,
OZAKI,
AND
TAKEICHI
Preparation of trypsin-inhibitor affinity column. In order to prepare an affinity column of trypsin inhibitor, 10 mg of soybean trypsin inhibitor was coupled with 1 g of CNBr-activated Sepharose 4B (Pharmacia) following the method recommended by the company. One milliliter of coupled gel was packed in a 2-ml plastic syringe. The capacity of this trypsin-inhibitor column was large enough to be used for the present purpose, as checked by assaying the activity of trypsin bound to it by a spectrophotometric method (Schwert and Takenaka, 1955). RESULTS
Preparation of V79 Cells with only Ca2+independent Adhesion Mechanism In order to determine the critical concentration of trypsin to inactivate the Ca’+independent adhesion mechanism, cell monolayers were treated with various concentrations of trypsin in the presence of either 1 m&f EDTA or 1 mJ4 Ca2+ at 37°C for 15 min. The aggregating ability of these cells was assayed in the Ca2+-free medium (HCMF) to selectively detect the Ca2+-independent process of cell adhesion. As shown in Fig. 1, curve A, trypsin at concentrations higher than 0.01% was required to make cells completely nonadhesive in Ca*‘free conditions. Whether EDTA or Ca2+ was added to the trypsin solution did not affect the trypsin sensitivity of the Ca2’independent adhesion property of cells, as suggested previously (Takeichi, 1977). The trypsin sensitivity of the Ca2+-dependent mechanism cannot be assayed in the same system as above, because the Ca2+-independent mechanism which also operates in the aggregation of cells interferes with the detection of the Ca’+-dependent adhesion process. Therefore, cells having only the Ca2+-dependent adhesion property (TC-V79) were prepared first by the trypsin-Ca2+ treatment of Takeichi (1977). Then these cells were retreated with various concentrations of trypsin in the presence of EDTA or EGTA, to determine the
Cell Adhesion
Mechanisms
209
Trypsin mm. (‘1. I
FIG. 1. Trypsin sensitivity of the adhesive properties of V79 cells. (A) Aggregation of cells dispersed with trypsin at the concentrations indicated on the abscissa in the presence of 1 mIi4 EDTA. (B) Aggregation of cells which were once dispersed with the trypsin-Ca*+ treatment and received a retreatment with trypsin (at concentrations indicated on the abscissa) plus 1 m&f EDTA. The method of retreatment with trypsin was the same as that described in footnote a to Table 2. Both in (A) and (B), 1 x 10’ cells were treated with 5 ml of trypsin solution. Aggregation was assayed after 2 hr of gyration in Ca’+-free aggregation medium (A) or in Ca*’ (1 n&f)-containing medium (B).
critical concentration of trypsin for inactivation of the Ca2+-dependent adhesion mechanism. As shown by curve B in Fig. 1, the Ca”-dependent adhesive property of cells is very sensitive to trypsin; 0.0001% of trypsin was enough to remove this adhesion property from cells. If Ca2+ (1 mJ4) was added to the trypsin solution, Ca2+-dependent adhesiveness was not inactivated at all at any concentration of trypsin tested. As EDTA had the same effects as EGTA, the former chelater was used in the following experiments described-in this report. To summarize, the critical concentration of trypsin needed to abolish the Ca2+-independent adhesion property is O.Ol%, and that needed to abolish the Ca2+-dependent adhesion property is 0.0001% in Ca2+-free medium. On the basis of these results, it can be predicted that cells dispersed with 0.0001% trypsin containing 1 mM EDTA (light trypsin-EDTA treatment) directly from cell monolayers do not have the Ca’+dependent mechanism of adhesion but retain the Ca2’-independent one. Figure 2 shows the aggregation kinetics of V79 cells
210
DEVELOPMENTALBIOLOGY
dispersed with this light trypsin-EDTA treatment (LTE-V79). As expected, aggregation of these cells was completely Ca’+independent. These cells aggregated also at a low temperature (4’33, which is characteristic of aggregation by the Ca2+-independent mechanism (Takeichi, 1977). In Table 1, the aggregation properties of V79 cells dispersed with four different treatments, three of which had already been published (Takeichi, 1977), were summarized. We postulated from previous work (Takeichi, 1977) that E-V79 have both the Ca2+-dependent and Ca”-independent mechanisms, TC-V79 have only the Ca*+dependent mechanism, and TE-V79 have neither one. LTE-V79 are assumed to have only the Ca2’-independent mechanism from the above results. To reconfirm these
VOLUME 70,1979
assumptions, the following experiments were performed. If our assumptions are correct, cells with only the Ca2+-dependent mechanism of adhesion or cells with only the Ca2’-independent mechanism should be obtained from E-V79 by the appropriate treatment with trypsin. As shown in Table 2, E-V79 retreated with “trypsin-Ca”” aggregated TABLE 2 EFFECTOF TRYPSIN TREATMENTSON ADHESIVE PROPERTYOF ONCE-DISPERSEDV79 CELLS Cells Second treatment” Aggregation treated WdNo) *
E-V79
TC-V79 LTE-V79
-Co’+ I 0
1 30 Time
I
I
60
120 lmln)
FIG. 2. Aggregation of V79 cells dispersed with O.OOOl% trypsin + 1 mMEDTA (LTE-V79) in medium either with 1 mM Ca’+ (0) or without Ca*+ (0).
Trypsin-Ca*’ Light trypsin-EDTA EDTA Light trypsin-EDTA Trypsin-Ca2+ Trypsin-Ca2+ Light trypsin-EDTA
With Ca2+
With. out t-h=+ --
0.325 0.301 0.211 0.890 0.186 0.995 0.285
0.998 0.307 0.350 0.837 l.OOO 0.992 0.273
“Cells once dispersed by appropriate treatments were collected in a centrifuge tube and incubated with the reagents for the second treatment at 37°C for 15 min. In the case of trypsin-Ca*+ treatment, cells were incubated in 1 mM Ca” for 10 min prior to the second treatment. Retreated cells were washed in the same way as the first treatment and finally suspended in HCMF. Trypsin-Ca2+ = 0.01% trypsin + 1 mM Ca*+; light trypsin-EDTA = O.OOOl%trypsin + 1 mM EDTA; EDTA = 1 mM EDTA. Cells which received a second treatment with trypsin-EDTA (0.01%trypsin + 1 mM EDTA) lost their adhesiveness completely in every case. b Shown by aggregation index after a 2-hr incubation.
TABLE 1 ADHESIVE PROPERTIESOF V79 CELLS DISPERSEDWITH DIFFERENT TREATMENTS Abbreviated cell Presumed adhesion mechanisms Dispersion treatment” Aggregation* intact” name With Ca*+ Wg-rgut E-V79 TC-V79 LTE-V79 TE-V79
EDTA Trypsin-Ca*+ Light trypsin-EDTA Trypsin-EDTA
+++ +++ +++ -
-i--l+++
Ca”-dep and Ca”-indep Ca’+-dep Ca”-indep None
a EDTA = 1 mM EDTA; Trypsin-Ca2+ = 0.01%trypsin + 1 m&f Ca’+; light trypsin-EDTA + 1 mM EDTA; trypsin-EDTA = 0.01% trypsin + 1 mlM EDTA. ’ Data for E-, TC-, and TE-V79 are from Takeichi (1977). ’ Ca”-dep = Ca”-dependent mechanism; Ca2+-mdep = Ca*+-independent mechanism.
= O.OOOl% trypsin
URUSHIHARA,
OZAKI,
AND
Cell Adhesion
TAKEICHI
centration of Fab, although less than in the control, as shown in Fig. 3a. The Fab strongly inhibited the aggregation of LTEV79 both in the presence and absence of Ca2+. The aggregation of TC-V79, however, was not affected at all (Figs. 3c and 3b). The inhibitory effect of Fab was dependent on its concentration as shown in Fig. 4, the
only in the presence of Ca”, while E-V79 retreated with “light trypsin-EDTA” aggregated to the same degree in the presence and absence of Ca2+. As is also shown in Table 2, TC-V79 retreated with “light trypsin-EDTA” and LTE-V79 retreated with “trypsin-Ca’+” lost their adhesiveness completely. These results are totally consistent with the above assumptions on the distribution of two adhesion mechanisms in cells dispersed with different treatments as shown in Table 1. The second trypsin treatment in these experiments did not increase the dead cell population significantly, as judged from growing ability of the cells which received the second treatment in normal culture medium. Usually, more than 90% of the cells were alive at the end of the above experiments.
0
0 001
0 01 Fab
Inhibition of Vi’9 Cell Aggregation by Fab Fragments of Anti-E-V79 Antibody
01 con
