Cell, Vol. 18, 391-398,
October
1979,
Copyright
0 1979
h Involved
in Cell Aggregation
John E. Geltosky, James Weseman, Antony and Richard A. Lerner Department of Cellular and Developmental Immunology Research Institute of Scripps Clinic La Jolla, California 92037
by MIT
Bakke
Summary Analysis of the composition of cell surface-associated glycoproteins of D. discoideum by lactoperoxidasecatalyzed radioiodination, followed by isolation by Con A-Sepharose chromatography, revealed that the developmentally regulated cell surface expression of a certain glycoprotein (gp150) parallels the onset of mutual cellular cohesiveness (Geltosky, Siu and Lerner, 1976). We have purified gpl50 and raised specific antibodies to it. Through utilization of the specific antibody and a fluorescence-activated cell sorter, the expression of gpl50 on the cell surface has been studied. Starting from a low level in noncohesive (vegetative) cells, there is a rapid accumulation of gpl50 on the surfaces of aggregating cells. A peak level of expression is achieved by 10 hr and maintained at least until the steps of terminal differentiation. Most significantly, monovalent Fab’ derived from anti-gp150, when added to aggregation-competent cells, blocks the cells’ ability to reaggregate. Fab’s derived from antisera with different specificities were ineffective inhibitors of cell aggregation. These results suggest that gpl50 serves an intimate role in cell adhesion. Introduction Dictyostelium discoideum, when provided with an adequate supply of nutrients, exists as independent amebae; upon starvation the cells undergo chemotaxis toward CAMP, display a mutual cellular cohesiveness and finally aggregate into discrete pseudoplasmodia, or slugs. Further morphogenetic events result in the formation of a mature fruiting body, consisting of spores encapsulated in a sporangium, supported by a stalk. It is the well defined, highly synchronous manner in which the cells come together into aggregates that we and other investigators have utilized to investigate the mechanisms of cellular cohesion. As a point of departure, we investigated the possible role which cell surface-associated glycoproteins may play in mediating cell aggregation. Glycoproteins, as a class of molecules, have been implicated as serving roles in a number of cell surface events (Oseroff, Robbins and Burger, 1973; Thiery et al., 1977; Wylie, Damsly and Buck, 1979). Cell cohesion in D. discoi-
in D. discoideum deum appears in part to involve cell surface-associated lectins (Rosen et al., 1973; Rosen, Reitherman and Barondes, 1975; Siu et al., 1976) as well as certain plasma membrane-associated glycoproteins (Hoffman and McMahon, 1978; Muller and Gerisch, 1978). Gerisch and his colleagues, using serological methods, described the existence of a set of cell surface antigens unique to aggregation-competent cells in D. discoideum. These antigens were given the operational term contact site A. Monovalent Fab’s derived from this antiserum made specific for contact site A by absorption against vegetative cells blocked the ability of the cells to self-aggregate. Hence contact site A was thought to contain a molecule(s) crucial to cell aggregation. Recently, contact site A has been purified and shown to consist of a single glycoprotein with a molecular weight of 80,000. As part of our studies, we previously described the developmentally regulated expression on the cell surface of a glycoprotein of approximate molecular weight 150,000 (gpl50) (Geltosky, Siu and Lerner, 1976). This protein is expressed at a low level in vegetative cells, and its concentration on the cell surface becomes markedly enhanced during the period of mutual cellular cohesiveness. To assess rigorously the possible role that gpl50 may play in cell adhesion mechanisms, we have purified the molecule and raised antibodies to it. In this paper, we describe the kinetics of expression of gpl50 on the cell surface and show that monovalent Fab’ fragments directed to gpl50, when reacted with aggregation-competent cells, block the ability of these cells to reaggregate. These results imply a crucial role for gpl50 in cellular adhesion of D. discoideum. Results Isolation of gpl50 Since the developmentally regulated expression of gpl50 on the cell surface strongly correlates with the onset of mutual cellular cohesiveness, we felt that this molecule might be actively involved in the cell aggregation process. With this in mind, starting from large amounts of total cellular Con A binding protein, we isolated gpl50 by preparative SDS-PAGE, as detailed in Experimental Procedures. From 5 x 10” 16 hr cells, 25 mg of total cellular glycoprotein were routinely recovered, representing an approximate enrichment of 60 fold. Figure 1 depicts the population of total cellular Con A binding proteins (lane A), which appears qualitatively very similar to those glycoproteins detected by cell surface radioiodination, followed by Con A-Sepharose chromatography (data not shown). Reelectrophoresis of the material eluted from that region of the gel corresponding in mobility to
Cell 392
MWxlO-3
MWx103
150130705645-
70-
C Figure teins
5645-
1. Cell Surface
of Total
Cellular
Con A Binding
Pro-
(A) Total cellular Con A binding proteins radioiodinated by the chloramine T procedure (McConahey and Dixon, 1966); (6) immunoprecipitation by antigpl50 gammaglobulin-Sepharose. Anti-gpl50 gammaglobulin coupled to Sepharose was incubated with 1 x 10’ cpm of radioiodinated Con A binding proteins and treated as described in Experimental Procedures; (C) immunoprecipitation with preimmune gammaglobulin-Sepharose. Preimmune gammaglobulin coupled to Sepharose was reacted with ‘251-Con A binding proteins and treated as in lane B. The material at the bottom of lane S is Con A that was present in the antigen mixture.
A Figure
2. lmmunoprecipitation
Con A Binding
B
Proteins
(A) Coomassie brilliant blue stain of total cellular Con A binding proteins isolated as described in Experimental Procedures; (6) Coomassie brilliant blue stain of purified gpl50. The material in the 5065K region of the gel is a contaminant in the acrylamide and should be ignored.
gpl50 establishes the fact that the material is homogeneous (lane B). It is this material that is injected into rabbits. Characterization of Antisera to gpl5D In order to assess the specificity of the antisera to electrophoretically homogeneous gpl50, immunoprecipitation of total cellular Con A binding protein was used (Figure 2, lane B). In this case, lz51 glycoproteins were treated with anti-gpl50 gammaglobulin coupled to Sepharose beads as outlined in Experimental Procedures. A very marked enrichment for gp150 ac-
trues from this treatment; the minor amount of extraneous bands seen in the gel represents nonspecifically adsorbed material, most of which is detected when preimmune gammaglobulin coupled to Sepharose is used in place of immune globulin (Figure 2, lane 0. Occasionally we detect low levels of two other glycoproteins, gp70 and gpl40, in the anti-gpl50derived precipitates. The reason for this is currently under investigation. Expression of gpl5D on the Cell Surface during Development To demonstrate binding of anti-gpl50 to the cell surface, indirect immunofluorescence studies were carried out (Figure 3). Developed cells treated with antigp150 serum display intense fluorescence over the entire cell surface; cells treated with a number of different preimmune sera as the first antibody do not display any surface fluorescence. The FACS II was used to quantitate the expression of gp150 on the cell surface during development. Utilization of this technology also allowed us to study
Cell Surface 393
Glycoprotein
in D. discoideum
stalk and spore cells, and this differential gpl50 expression may partition into the two populations. This possibility is being investigated. A mean can be calculated for each histogram, and it is seen to vary as a function of time in development (Figure 5). The ordinate of this figure expresses the concentration of gpl50 per unit of surface area across the cell surface. The size of these cells, as indicated by light scatter data, decreases 2-fold between 0 and 18 hr of development. During development there is a rapid increase in the concentration of gpl50 on the cell surface, with a plateau which occurs at roughly the 10 hr point and which is maintained until 18 hr. This amount of fluorescence corresponds to 5.5 x 1 O6 molecules of anti-gpl50-Fab’ bound to the surface of an 18 hr cell (Experimental Procedures).
Figure 3. Indirect lmmunofluorescence face Binding by Anti-gpl50
Demonstration
of Cell Sur-
16 hr cells (2 x IO’) were dissociated and treated with 50 ~1 of a 1 / 40 dilution of anti-gp150 serum made in DS. Following a 30 min incubation at room temperature, the cells were extensively washed with cold DS. The cells were then treated with 50 ~1 of rhodamineconjugated goat anti-rabbit (Cappel Laboratories) for 30 min at room temperature. Following extensive washes in cold DS, the cells were then viewed with fluorescence optics. When preimmune serum is used as the first antibody, no labeling is detected. Magnification 400x.
the homogeneity of a cell population with regard to expression of gpl50 on the cell surface. Figure 4 illustrates the fluorescence histograms generated for cells derived at different times in the developmental sequence. Each point on the curve represents the number of cells having that intensity of fluorescence. A saturation curve was first established using varying concentrations of Fab’. 100% of the cells were stained at the saturating concentration (3.5 mg/ml). The histograms derived for vegetative, 6 hr and 12 hr cells are reasonably monomodal, indicating a homogeneous population of cells expressing gpl50 on their cell surfaces; however, the histogram derived for 18 hr cells is apparently bimodal (peaks roughly at channel numbers 200 and 300), indicating two populations of cells distinguished by their abilities to express gpl50 at the cell surface. It is at this point in development that cells begin to undergo terminal differentiation into
Effect of Fab’ to gp150 on Cell Aggregation If gpl50 were intimately involved in the mechanics of cell aggregation, one would expect that specific coating of the molecule with monovalent Fab’ would abrogate its ability to interact with neighboring cells in a cognitive fashion, thereby resulting in a blockage of cell aggregation. This strategy has been used in a number of systems (Beug, Katz and Gerisch, 1973; Rosen, Chang and Barondes, 1977; Brackenbury et al., 1977). We dissociated 15 hr cells into a single cell suspension, treated them with either Fab’ derived from anti-gpl50 or Fab’ from preimmune animals (or bovine serum albumin) and allowed the cells to reaggregate. The extent of aggregation in these experiments was determined by measuring the absence of single cells as they were being recruited into multicellular aggregates. Figure 6 visually demonstrates these events: at the onset of the incubation, the cells are completely dissociated (A); within 10 min, large multicellular aggregates appear, while the number of single cells is severely reduced (B); when the incubation mixture contains anti-gpl50-Fab’ at a final concentration of 0.6 mg/ml, the cells remain largely dissociated, even up to 1 hr of incubation (C). If the ability of anti-gpl50Fab’ to block cell aggregation is assayed as a function of Fab’ concentration, the curve in Figure 7 is derived. Complete blockage of aggregation essentially occurs at a final anti-gpl50-Fab’ concentration of 0.6 mg/ ml. Three different preimmune Fab’ preparations (including that from the rabbit eventually used as a source of anti-gpl50-Fab’) were tested, and all were found to be without effect on the aggregation process. Specificity of Immune Inhibition of Cell Aggregation In an attempt to delineate further the specificity of the blockage of cell aggregation by anti-gpl50-Fab’, we tested two other antisera with different immunospecificities: one was derived to gpl30. a cell sut-faceassociated glycoprotein whose expression is not under developmental regulation; the other was raised to
Cell 394
Figure 4. Fluorescence Histograms of Cells Taken at Various Times in Development and Treated with Antigpl50-FITC-Fab’ Cells were taken at various points in development, treated with a saturating amount of antigpl50-FITC-Fab’ and analyzed by an FACS II cell sorter as described in Experimental Procedures. Each point on the curve represents a number of cells with a certain intensity. Note that the distributions for 0. 6 and 12 hr cells are monomodal; however, 16 hr cells display a bimodal distribution with respect to gpl50 expression at the cell surface (peaks at roughly channel numbers 200 and 300).
intact vegetative ceils. The specificity of each serum, when tested by immunoprecipitation of total Con A binding proteins, is depicted in Figure 8A. As noted before, anti-gpl50 specifically recognizes gpl50 (lane l), the extraneous bands also being present in the preimmune control (lane 4). Similarly, immunoprecipitation with anti-gpl30 results in a substantial enrichment for gp130 (lane 2). It also appears that the level of gp70 is enhanced in these precipitates, which we occasionally detect in anti-gpl50-derived precipitates. Anti-vegetative cell serum recognizes gp70, gpl30 and gpl50 in these immunoprecipitation analyses (lane 3). The extraneous bands detected in these precipitates, which are also present in the preimmune situation, do not represent specific interaction between antigen and antibody. Preimmune serum does not label the cell surface in either direct or indirect immunofluorescence studies, nor do Fab’s from preimmune sera affect cell aggregation. Since effective blockade of cell aggregation results from the binding of the Fab’ to the cell surface, we first quantitated the binding abilities of the various Fab’s to the surfaces of cells allowed to develop for 16 hr (Experimental Procedures). Cells treated in such a way bound virtually equivalent levels of all three FITC-Fab’s tested (Figure 88). Each Fab’ was then tested for its ability to block cell aggregation. Since all three Fab’s bound with equal efficiency to the cells, a single concentration of Fab’, which was the level that causes complete inhibition of aggregation by anti-gpl50-Fab’, was tested. When the abilities of anti-gpl30-Fab’ and anti-vegetative cell Fab’ were measured relative to anti-gp150 to block cell aggregation, it was found that the former was 1 1 %, the latter 22% as effective as anti-gpl50Fab’ in this regard. It is worth noting that anti-vegetative cell serum does precipitate some gp150; this
OJ Figure 5. Expression Time in Development
,
I
10 15 5 Time in Oevelopement [hrs] of gpl50
on the Cell Surface
20
as a Function
of
Means were calculated from both the fluorescent histograms shown in Figure 4 and histograms derived from light scatter data on the same cells (data not shown). By dividing the scatter mean into the fluorescent mean (ordinate), the binding is expressed as relative fluorescence intensity per unit area.
activity, no doubt, contributes some blockage activity in the cell aggregation assay. These results demonstrate that although all three antisera bind with equal efficiency to the surface of a cell allowed to develop for 16 hr, only the antiserum with specificity to gpl50 is effective in preventing cell aggregation. Discussion Our initial observation that the appearance on the cell surface is a developmentally
of gpl50 regulated
Cell Surface 395
Figure
Glycoprotein
6. Aggregation
in D. discoideum
of 16 Hr Cells in the Presence
of Anti-gpl
BO-Fab’
16 hr cells were harvested, dissociated into single cells and treated with Fab’ as described in Experimental Procedures. (A) Fully dissociated 16 hr cells at the start of the incubation; (B) cells after 10 min of incubation in the presence of 0.6 mg/ml preimmune Fab’; note the large multicellular aggregates and the small number of single cells; (C) cells after 10 min of incubation in the presence of 0.6 mg/ml anti-gpl50-Fab’. Phase optics were utilized. Magnification 100X.
event, and that this expression closely matches the onset of mutual cellular cohesiveness, suggested that this molecule may be intimately involved in the mechanics of cell aggregation of D. discoideum. The data in the present report substantiate this notion; however, a number of caveats must still be considered. It is possible that the interaction between a particular Fab’ and its antigen could result in a selective steric impairment of another set of molecules, those which are authentically involved in the mechanics of cell adhesion. Second, the molecule under study may exert a controlling or modulating influence on other plasma membrane-associated molecules, and perturbation of its function could therefore have pleiotropic consequences. Perhaps a more fundamental concern in these types of experiments is the biological relevance of the cell aggregation events measured in vitro. It is of interest to compare these findings on the involvement of gpl50 in cell aggregation in Dictyostelium with two other systems which have undergone detailed study. A large cell surface-associated proteoglycan complex has been shown to be involved in mediating cell cohesion in the sponge (Henkart, Humphreys and Humphrey& 1973). In this highly specialized case, aggregation of cells apparently occurs as a result of the interaction between an extracellular matrix consisting of this proteoglycan complex and cell surface-associated receptors (Muller et al., 1978). The overall process is species-specific. The second
0
.i
.i
.i
.i
.!I .6 .i
.i
.3
1:o
111
1:2
W’l IWmll Figure 7. Aggregation of 16 Hr Cells Concentrations of Anti-gpl50-Fab’
in the Presence
of Varying
16 hr cells were dissociated and treated with varying amounts of antigpl50-Fab’. Aggregation was allowed to proceed for 30 min. and the number of single cells remaining was determined by the particle counter as described in Experimental Procedures. Similar incubations were set up using Fab’ from a preimmune serum at two different concentrations (0.5 and 2 mg/ml); the number of single cells left after 30 min in both cases was identical and used to generate the zero concentration point on the graph.
system which has undergone extensive investigation is the neural retina cells of the chick embryo. Aggregation between these cells is due, in part, to the action of a cell surface-associated glycoprotein of molecular weight 140,000 daltons (Thiery et al., 1977). Treat-
Cell 396
Immunospecificity
A.
Figure 8. Specificity of Block of Cell Aggregation
Anti-gpl
BO-Fab’
MWws3
(A) lmmunoprecipitation of “‘1 total cellular Con A binding protein. Approximately 1 x 10’ cpm of ‘251-Con A binding protein from 16 hr 150cells were reacted with an appropriate dilution of each antiserum as described in Experimen130tal Procedures. Immune complexes were retrieved with S. aureus; following three washes 70in a-mm in TN, the pellets were boiled in electrophoresis sample buffer and subjected 56 to SDS-PAGE, and an autoradiogram was derived from each lane. (Lane 1) Immunoprecip45' itation with anti-gpl50; 10 ~1 of i/40 dilution of antiserum were used: note that the extraneous bands present here are also present in the preimmune incubations (lane 4). (Lane 2) lmmunoprecipitation with anti-gpl30; 30 pl of l/20 dilution of antigpl30 serum were used to generate this lane; note the enhanced reactivity for gp130 in this case over the other B. Amount of PITC-Fab' Bound at Saturation lanes in the figure. (Lane 3) ImmunoprecipitaRelative Fluorescence tion with anti-vegetative cell; 30 AI of a l/20 Fab' Units dilution were used in this case; when tested anti-gpl50 100 with this antigen (whole cell Con A binding anti-gp130 113 proteins). the antiserum reacts with gpl50. anti-vegetative cell 121 gpl30 and gp70. (Lane 4) Immunoprecipitation with preimmune serum: 10 f.rI of a l/20 C. Ability of Various Fab"s to Block Cell Aggregation dilution of a preimmune serum were used; a number of different preimmune sera gave proNumber of X Inhibitiion Relative files similar to this one. R Pab' Single Cells to Anti-gpl50-Pab' x (B) Saturation binding of FITC-Fab’ to 16 hr cells. Dissociated 16 hr cells were treated with 1.840 100.0 anti-gp150 15,184 6,464 .207 11.3 anti-gp130 the appropriate FITC-Fab’. and the amount of cell 7,755 .440 24.0 anti-vegetative fluorescence bound to the cell was quantitated 5,354 0 0 pre-imune as described in Experimental Procedures. The Fab’s had virtually identical FITC to protein ratios, so that these numbers are directly comparable. These data represent the averages of two separate experiments, where duplicate samples were run for each separate Fab’. (C) Ability of Fab’s of various specificities to block cell aggregation. Cells allowed to develop for 16 hr were dissociated into a single cell suspension and reacted with the appropriate Fab’, each at a fixed concentration of 0.7 mg/ml. Afler 30 min of shaking at 25°C. the extent of aggregation was measured with a particle counter, as outlined in Experimental Procedures. For these experiments, an index of inhibition of aggregation (R.) has been defined: R. =
number
of single cells after incubation number
in X-number
To compare directly the effect of the various the particular Fab’ relative to that of anti-gpl50 Rx R anti-gpl50
of single
of single cells afler incubation
cells after incubation in preimmune
Fab’s on cell aggregation is derived as follows:
relative
in preimmune
Fab’
Fab’ to the effect
by anti-gpl50-Fab’.
a percentage
of inhibition
by
x 100
ment of cells with Fab’ fragments rendered specific to this molecule blocks cell aggregation. The means by which this molecule mediates cell cohesion remains to be elucidated. The relationship between gpl50 and contact site A, which is the molecule(s) ascribed by Gerisch and coworkers to be responsible for cell aggregation in D. discoideum, is unclear. Recently, contact site A has been isolated and shown to consist of a single glycoprotein of approximate molecular weight 80,000 daltons (Huesgen and Gerisch, 1975). Thus contact site A and gpl50 do not appear to be the same molecule; however, the two molecules could still be antigenically related and the difference in size could be due to glycosylation. In an attempt to answer this question,
we derived a preparation enriched in contact site A (Huesgen and Gerisch, 1975). When this material was radioiodinated and immunoprecipitated in the standard fashion with anti-gpl50, we did not detect a band in the 80,000 dalton region of the gel. It therefore appears that gpl50 and contact site A are unrelated molecules. A mass of evidence has been presented which implicates a cell surface-associated carbohydrate binding protein as serving a crucial role in slime mold aggregation (Rosen et al., 1973, 1975; Siu et al., 1978; Ray, Shinnick and Lerner, 1979). The relationship, if any, between gpl50 and this protein remains to be studied. It is certainly possible, and perhaps likely, that cell aggregation is a consequence of a
Cell Surface 397
Glycoprotein
in D. discoideum
number of molecular systems operating at the cell surface, and that contact site A, carbohydrate binding protein and gpl50 belong to different systems. Experimental
Procedures
Cell Growth The wild-type strain NC-4 was used solely in these studies. Conditions for cell growth, development and harvest are identical to those previously described (Geltosky et al., 1976). Preparation of Total Cellular Con A Binding Protein For large-scale preparative purposes, 5 X 10” 16 hr cells were harvested from pads and lysed in 0.5% NP40. 10 mM NaN3 and 0.01% phenylmethylsulfonylfluoride made up in 25 mM Tris. 150 mM NaCl (pH 7.5) (TN). The cells were further disrupted by ultrasonication in a 20KHz Branson sonifer S125 equipped with a one eighth inch probe, and the resulting material was centrifuged at 10,000 rpm for 15 min in a refrigerated Sorvall. The supernatant was then applied to a 5 ml Con A-Sepharose column containing 15 mg of ConA per ml settled beads. The affinity column was prepared as previously described (Geltosky et al., 1976). Following sample application. unbound material was washed off with TN at an approximate flow rate of 0.5 ml/min. When the output absorbance at 280 mp fell to 0.1, specific elution of the glycoproteins by 0.2 M a-methyl-D-mannoside (a-mm) in TN was initiated. The material corresponding to the absorbance peak was pooled, exhaustively dialyzed against HP0 and finally lyophilized. Isolation of gpls0 Approximately 2.5 mg of the above isolated total Con A binding protein was loaded onto a 7% acrylamide gel. To localize gpl50 on the gel without staining for protein, dansylated gammaglobulin was run in a neighboring lane. Conventional protein staining results in fixation of the molecules in the gel, a step we felt wise to avoid, since further loss of native antigenicity and difficulty in recovery of material would accrue. Native gammaglobulin (approximate molecular weight 155,000) has roughly the same electrophoretic mobility as gpl50. Dansylated gammaglobulin was visualized on the gel with the aid of an ultraviolet light: a strip (approximately 2 mm wide) was then excised from the gel corresponding to this location, and gpl50 was eluted from the acrylamide with water in an overnight incubation. This material was then lyophilized and subsequently used for immunization of rabbits. Preparation of Antisera, Gammaglobulin and Fab’ In order to generate antibodies to gpl50, approximately 60 gg of lyophilized gpl50 were mixed with incomplete Freund’s adjuvant and injected subcutaneously into a rabbit. This protocol was repeated fortnightly over a 6 week period. At this time, antibody activity to gpl50 was detected in the serum of the immunized animals. Thereafler. the animals received monthly boosts of gpl50. To obtain gpl30 for immunization purposes, total Con A binding protein was run on 7% SDS gels and gpl30 was visualized by Coomassie brilliant blue staining. A number of bands corresponding to gpl30 were excised from the gel, and the proteins were retrieved by electrophoresis of the isolated slices. Following dialysis and lyophilization, 50-60 pg were injected into a rabbit according to the same protocol as that used for gpl50. Anti-vegetative cell antiserum was generated by injecting approximately 1 X 10’ vegetative cells mixed in incomplete Freund’s adjuvant into a rabbit using the same injection schedule as above. The gammaglobulin fraction of serum was isolated by conventional means: 50% saturated ammonium sulfate precipitation. followed by elution from an anion-exchange resin. Monovalent Fab’ fragments were prepared according to established procedures with some modification (Nisonoff et al., 1960). Divalent F(ab’), fragments were prepared by treating gammaglobulin with pepsin (3% w/w) at pH 4.0 for 4 hr at 37°C. The digest was then dialyzed versus TN and
subjected to chromatography on Sephadex G-l 50 to yield divalent F(ab’), devoid of undigested gammaglobulin and remnants of Fc. Monovalent Fab’ was obtained by first reducing divalent F(ab’), in 0.02 M dithiothreitol for 2 hr at room temperature, followed by alkylation in 0.08 M iodoacetamide for 30 min at room temperature. Following dialysis versus 17 mM phosphate (pH 6.4)(PB). monovalent Fab’ was stored in aliquots at -2O’C. Intact gammaglobulin was not detected in these materials when tested by immunodiffusion or SDSPAGE. When monovalent Fab’ directed to gp150 was tested for antigen binding ability, either by immunoprecipitation or immunofluorescence, strong reactivity was detected. lmmunoprecipitation To test the specificity of the various antisera used in these experiments, immunoprecipitation of radioiodinated total cellular Con A binding protein was utilized. For routine analysis, the following protocol was used. Total Con A binding protein was radioiodinated by chloramine T oxidation to give an approximate specific activity of 3 x 1O5 cpm/pg of protein (McConahey and Dixon, 1966). For immunoprecipitation. the reaction mixture typically contained 8 X 1 O6 cpm of 1251glycoprotein, from lo-20 ~1 of antisera diluted from I:20 to 1: 50, and 100 pl of 0.1 M a-mm, 0.6 M NaCI. 10m4 M KI. and 25 mM Tris (pH 7.5). Mannoside was included in the incubations to negate any interactions between glycoprotein and small amounts of Con A which may have washed off the column during the preparation of the glycoproteins. The incubations were carried out overnight at 4°C; immune complexes were then collected with the addition of 25 ~1 of a suspension of formalin-treated Staphylococcus aureus (Kessler. 1975). Following a 30 min incubation at room temperature, S. aureus was collected by centrifugation and washed 3 times in 0.1 M a-mm, 0.6 M NaCI. 10m4 M KI and 25 mM Tris (pH 7.5). The final pellet was then boiled in electrophoresis sample buffer, the S. aureus was centrifuged out and the supernatant was subjected to SDS-PAGE. Following electrophoresis. the gel was dried down and an autoradiogram was derived through use of Kodak X-Omat RP film. A second method of immunoprecipitation analysis was also used occasionally. Gammaglobulin was prepared from immune serum and coupled to Sepharose to give a final concentration of 5 mg of gammaglobulin coupled per ml of packed Sepharose (Porath et al.. 1973). Approximately 1 x 10’ cpm of ‘251 Con A binding protein were added to 0.5 ml settled volume of anti-gpl50 gammaglobulin Sepharose and 1 ml of 0.1 M a-mm, 2 mg/ml bovine serum albumin in TN. This mixture sat at 37°C for 2 hr with occasional mixing. Following this incubation period, the beads were collected by centrifugation and washed 3 times in the above buffer. The final pellet of beads was treated with electrophoresis sample buffer for 1 hr at 37’C. Following this, the beads were centrifuged and the supernatant was subjected to SDS-PAGE. Electrophoresis Conditions Electrophoresis of proteins was carried out as previously described (Geltosky et al., 1976). 7% acrylamide gels were solely used. Direct lmmunofluorescence To carry out direct immunofluorescence of cells for the cell sorter experiments, the Fab’ was labeled with fluorescein isothiocyanate (FIT0 to yield 1.6 molecule of FITC per molecule of Fab’ (Clark and Sheppard, 1963). Approximately 2 X lo6 cells were labeled with FITC-Fab’ at 5 mg/ml in 20 mM EDTA in PB (DS) in a volume of 50 ~1 for 20 min at room temperature. The cells were washed 6 times in cold DS and kept on ice prior to cell sorter analysis. In order to obtain a quantitative estimate of the amount of fluoresceinated antibody bound to the cells, labeling was carried out as above. At the end of the washing procedure, the cells were lysed in 0.6 ml of 0.1 N NaOH. Fluorescence was measured in a Perkin-Elmer Model MPF-44A. using an excitation wavelength of 490 rnp and an emission wavelength of 530 mp. To insure linearity of response and also to derive the number of FITC-Fab’ molecules bound to the cell surface, a standard curve was constructed by adding known amounts of FITC-Fab’ to a cell extract prepared in 0.1 N NaOH.
Cdl 398
Flow Cytofluorimetry Cells labeled with FITC-conjugated antibody were analyzed by light scatter (to give cell size) and fluorescence on a Becton-Dickinson FACS II fluorescence-activated cell sorter. The laser was operated at 400 mW and the photomultiplier tube at 600 V. 5 c fluorescent beads (Coulter) were used to calibrate the machine.
Kessler, S. (1975). Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameter of the interaction of antibody antigen complexes with protein A. J. Immunol. 115. 1617-1624.
Cell Aggregation Assay The ability of the various monovalent Fab’s to block reaggregation of aggregation-competent cells was assayed by measuring the decrease in the number of single cells remaining after a given time of incubation. Cells allowed to develop for 16 hr were dissociated into a single cell suspension by repeated pipettings in DS. The cell concentration was adjusted to 1 x 1 07/ml and 50 ~1 were added to a 12 x 75 mm glass culture tube. An equal volume of Fab’ solution (made up in PB) was then added, and the tubes were allowed to sit on ice for 30 min. At the end of this time, the tubes were vortexed and then shaken at 150 rpm on a New Brunswick shaker (model Gl 0) maintained at 22’C. The aggregation reaction was terminated upon pipetting the entire reaction volume into 20 ml of 10 mM EDTA in PB. In general, triplicates were run for each determination. Single cells were counted in a Coulter Particle Counter (model 261) using the following settings: 1 /amplification = l/2, 1 /aperture current = 1, lower threshold = 10 and upper threshold = 60. Adherence to these conditions allowed 90% of the single cells to be counted. For each sample, two readings were made, each expending 0.5 ml of the total 20 ml. As a control for each experiment, incubations were carried out in the presence of either Fab’ from a preimmune rabbit or bovine serum albumin. Identical results were achieved with both these controls.
Mtiller. K. and Gerisch. G. (1978). A specific glycoprotein as the target site of adhesion blocking Fab in aggregating Dictyostelium cells. Nature 274, 445-449.
Acknowledgments We wish to thank Dr. Juan Yguerabide and Dr. Al Jesaitis of UCSD for providing us with the spectrofluorometer and advice concerning the fluorescence experiments. This research was supported in part by a research fellowship from the American Cancer Society. This is publication number 106 from the Department of Cellular and Developmental Immunology. and number 1763 from the Research Institute of Scripps Clinic, La Jolla, California. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
May
15, 1979;
revised
Beug. H.. Katz, F. E. and Gerisch, G. (1973). Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum. J. Cell Biol. 56, 647-658. Brackenbury. R.. Thiery. J.-P., Rutishauser. U. and Edelman, G. M. (1977). Adhesion among neural cells of the chick embryo. J. Biol. Chem. 252, 6835-6840.
Geltosky, J. E., Siu. C.-H. and Lerner. the plasma membrane of Dictyostelium ment. Cell 8. 391-396.
R. A. (1978). discoideum
Henkart. P., Humphreys. S. and Humphreys. zation of sponge aggregation factor. A unique Biochemistry 72, 3045-3050.
A method Int. Arch.
of trace iodinaAllergy Applied
Miller, W. E. G.. Zanv, R. K.. Kurelec. B. and Miller, I. (1978). Species-specific aggregation in sponges. Transfer of a species-specific aggregation receptor from Suberites domuncula to cells from Geodia cydonium. Differentiation 10, 55-60. Nisonoff, A., Wissler, F. C.. Lyman, L. N. and Woernley. D. L. (1960). Separation of univalent fragments from the bivalent antibody molecule by reduction of disulfide bonds. Arch. Biochem. Biophys. 89, 230244. Oseroff, A. R.. Robbins. membrane: biochemical Biochem. 42, 642-682.
P. W. and Burger, M. (1973). The cell surface aspects and biophysical probes. Ann. Rev.
Porath, J., Aspberg. K., Drevin, of cyanogen bromide-activated 86, 53-56.
H. and Axen. R. (1973). Preparation agarose gels. J. Cl?romatography
Ray, J., Shinnick. T. and Lerner. R. A. (1979). A mutation altering the function of a carbohydrate binding protein blocks cell-cell cohesion in developing Dictyostelium discoideum. Nature 279, 215-221, Rosen, S. D.. Chang, C.-M. and Barondes. S. H. (1977). Intercellular adhesion in the cellular slime mold Polyspondilium pallidurn inhibited by the interaction of asialofetuin or specific univalent antibody with endogenous cell surface lectin. Dev. Biol. 61, 202-213. Rosen, S. D.. Reitherman, R. W. and Barondes, S. H. (1975). Distinct lectin activities from six species of cellular slime molds. Exp. Cell Res. 95, 159-l 86. Rosen, S. D.. Kafka, J. A., Simpson, D. L. and Barondes, S. H. (1973). Developmentally regulated carbohydrate binding protein in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA 70. 25542558. Siu. C-H.. Lerner, R. A.. Ma, G.. Firtel. R. A. and Loomis, W. F. (1976). Developmentally regulated proteins of the plasma membrane of Dictyostelium discoideum. The carbohydrate-binding protein. J. Mol. Biol. 100, 157-l 78. Thiery, J.-P., Brackenbury. R.. Rutishauser. U. and Edelman, G. M. (I 977). Adhesion among neural cells of the chick embryo. II. Purification and characterization of a cell adhesion molecule from neural retina. J. Biol. Chem. 252, 6841-8845.
July 20, 1979
Clark, H. F. and Sheppard, C. C. (1963). A dialysis preparing fluorescent antibody. Virology 20, 642-644.
McConahey. P. J. and Dixon, F. J. (1966). tion of proteins for immunological studies. Immunol. 29, 185- 189.
technique
for
Glycoproteins of during develop-
T. (1973). Characteriproteoglycan complex.
Hoffman, S. and McMahon. D. (1978). Defective glycoproteins in the plasma membrane of an aggregation minus mutant of Dictyosteliom discoideum with abnormal cellular interactions. J. Biol. Chem. 253, 278-287. Huesgen. A. and Gerisch, G. (1975). Solubilized contact site A from cell membranes of Dictyostelium discoideum. FEBS Letters 56, 4649.
Wylie, D. E., Damsly. C. H. and Buck, C. A. (1979). Studies on the functions of cell surface glycoproteins I. Use of antisera to surface membranes in the identification of membrane components relevant to cell-substrate adhesion. J. Cell Biol. 80. 385-402.
October
1979,
Copyright
0 1979
h Involved
in Cell Aggregation
John E. Geltosky, James Weseman, Antony and Richard A. Lerner Department of Cellular and Developmental Immunology Research Institute of Scripps Clinic La Jolla, California 92037
by MIT
Bakke
Summary Analysis of the composition of cell surface-associated glycoproteins of D. discoideum by lactoperoxidasecatalyzed radioiodination, followed by isolation by Con A-Sepharose chromatography, revealed that the developmentally regulated cell surface expression of a certain glycoprotein (gp150) parallels the onset of mutual cellular cohesiveness (Geltosky, Siu and Lerner, 1976). We have purified gpl50 and raised specific antibodies to it. Through utilization of the specific antibody and a fluorescence-activated cell sorter, the expression of gpl50 on the cell surface has been studied. Starting from a low level in noncohesive (vegetative) cells, there is a rapid accumulation of gpl50 on the surfaces of aggregating cells. A peak level of expression is achieved by 10 hr and maintained at least until the steps of terminal differentiation. Most significantly, monovalent Fab’ derived from anti-gp150, when added to aggregation-competent cells, blocks the cells’ ability to reaggregate. Fab’s derived from antisera with different specificities were ineffective inhibitors of cell aggregation. These results suggest that gpl50 serves an intimate role in cell adhesion. Introduction Dictyostelium discoideum, when provided with an adequate supply of nutrients, exists as independent amebae; upon starvation the cells undergo chemotaxis toward CAMP, display a mutual cellular cohesiveness and finally aggregate into discrete pseudoplasmodia, or slugs. Further morphogenetic events result in the formation of a mature fruiting body, consisting of spores encapsulated in a sporangium, supported by a stalk. It is the well defined, highly synchronous manner in which the cells come together into aggregates that we and other investigators have utilized to investigate the mechanisms of cellular cohesion. As a point of departure, we investigated the possible role which cell surface-associated glycoproteins may play in mediating cell aggregation. Glycoproteins, as a class of molecules, have been implicated as serving roles in a number of cell surface events (Oseroff, Robbins and Burger, 1973; Thiery et al., 1977; Wylie, Damsly and Buck, 1979). Cell cohesion in D. discoi-
in D. discoideum deum appears in part to involve cell surface-associated lectins (Rosen et al., 1973; Rosen, Reitherman and Barondes, 1975; Siu et al., 1976) as well as certain plasma membrane-associated glycoproteins (Hoffman and McMahon, 1978; Muller and Gerisch, 1978). Gerisch and his colleagues, using serological methods, described the existence of a set of cell surface antigens unique to aggregation-competent cells in D. discoideum. These antigens were given the operational term contact site A. Monovalent Fab’s derived from this antiserum made specific for contact site A by absorption against vegetative cells blocked the ability of the cells to self-aggregate. Hence contact site A was thought to contain a molecule(s) crucial to cell aggregation. Recently, contact site A has been purified and shown to consist of a single glycoprotein with a molecular weight of 80,000. As part of our studies, we previously described the developmentally regulated expression on the cell surface of a glycoprotein of approximate molecular weight 150,000 (gpl50) (Geltosky, Siu and Lerner, 1976). This protein is expressed at a low level in vegetative cells, and its concentration on the cell surface becomes markedly enhanced during the period of mutual cellular cohesiveness. To assess rigorously the possible role that gpl50 may play in cell adhesion mechanisms, we have purified the molecule and raised antibodies to it. In this paper, we describe the kinetics of expression of gpl50 on the cell surface and show that monovalent Fab’ fragments directed to gpl50, when reacted with aggregation-competent cells, block the ability of these cells to reaggregate. These results imply a crucial role for gpl50 in cellular adhesion of D. discoideum. Results Isolation of gpl50 Since the developmentally regulated expression of gpl50 on the cell surface strongly correlates with the onset of mutual cellular cohesiveness, we felt that this molecule might be actively involved in the cell aggregation process. With this in mind, starting from large amounts of total cellular Con A binding protein, we isolated gpl50 by preparative SDS-PAGE, as detailed in Experimental Procedures. From 5 x 10” 16 hr cells, 25 mg of total cellular glycoprotein were routinely recovered, representing an approximate enrichment of 60 fold. Figure 1 depicts the population of total cellular Con A binding proteins (lane A), which appears qualitatively very similar to those glycoproteins detected by cell surface radioiodination, followed by Con A-Sepharose chromatography (data not shown). Reelectrophoresis of the material eluted from that region of the gel corresponding in mobility to
Cell 392
MWxlO-3
MWx103
150130705645-
70-
C Figure teins
5645-
1. Cell Surface
of Total
Cellular
Con A Binding
Pro-
(A) Total cellular Con A binding proteins radioiodinated by the chloramine T procedure (McConahey and Dixon, 1966); (6) immunoprecipitation by antigpl50 gammaglobulin-Sepharose. Anti-gpl50 gammaglobulin coupled to Sepharose was incubated with 1 x 10’ cpm of radioiodinated Con A binding proteins and treated as described in Experimental Procedures; (C) immunoprecipitation with preimmune gammaglobulin-Sepharose. Preimmune gammaglobulin coupled to Sepharose was reacted with ‘251-Con A binding proteins and treated as in lane B. The material at the bottom of lane S is Con A that was present in the antigen mixture.
A Figure
2. lmmunoprecipitation
Con A Binding
B
Proteins
(A) Coomassie brilliant blue stain of total cellular Con A binding proteins isolated as described in Experimental Procedures; (6) Coomassie brilliant blue stain of purified gpl50. The material in the 5065K region of the gel is a contaminant in the acrylamide and should be ignored.
gpl50 establishes the fact that the material is homogeneous (lane B). It is this material that is injected into rabbits. Characterization of Antisera to gpl5D In order to assess the specificity of the antisera to electrophoretically homogeneous gpl50, immunoprecipitation of total cellular Con A binding protein was used (Figure 2, lane B). In this case, lz51 glycoproteins were treated with anti-gpl50 gammaglobulin coupled to Sepharose beads as outlined in Experimental Procedures. A very marked enrichment for gp150 ac-
trues from this treatment; the minor amount of extraneous bands seen in the gel represents nonspecifically adsorbed material, most of which is detected when preimmune gammaglobulin coupled to Sepharose is used in place of immune globulin (Figure 2, lane 0. Occasionally we detect low levels of two other glycoproteins, gp70 and gpl40, in the anti-gpl50derived precipitates. The reason for this is currently under investigation. Expression of gpl5D on the Cell Surface during Development To demonstrate binding of anti-gpl50 to the cell surface, indirect immunofluorescence studies were carried out (Figure 3). Developed cells treated with antigp150 serum display intense fluorescence over the entire cell surface; cells treated with a number of different preimmune sera as the first antibody do not display any surface fluorescence. The FACS II was used to quantitate the expression of gp150 on the cell surface during development. Utilization of this technology also allowed us to study
Cell Surface 393
Glycoprotein
in D. discoideum
stalk and spore cells, and this differential gpl50 expression may partition into the two populations. This possibility is being investigated. A mean can be calculated for each histogram, and it is seen to vary as a function of time in development (Figure 5). The ordinate of this figure expresses the concentration of gpl50 per unit of surface area across the cell surface. The size of these cells, as indicated by light scatter data, decreases 2-fold between 0 and 18 hr of development. During development there is a rapid increase in the concentration of gpl50 on the cell surface, with a plateau which occurs at roughly the 10 hr point and which is maintained until 18 hr. This amount of fluorescence corresponds to 5.5 x 1 O6 molecules of anti-gpl50-Fab’ bound to the surface of an 18 hr cell (Experimental Procedures).
Figure 3. Indirect lmmunofluorescence face Binding by Anti-gpl50
Demonstration
of Cell Sur-
16 hr cells (2 x IO’) were dissociated and treated with 50 ~1 of a 1 / 40 dilution of anti-gp150 serum made in DS. Following a 30 min incubation at room temperature, the cells were extensively washed with cold DS. The cells were then treated with 50 ~1 of rhodamineconjugated goat anti-rabbit (Cappel Laboratories) for 30 min at room temperature. Following extensive washes in cold DS, the cells were then viewed with fluorescence optics. When preimmune serum is used as the first antibody, no labeling is detected. Magnification 400x.
the homogeneity of a cell population with regard to expression of gpl50 on the cell surface. Figure 4 illustrates the fluorescence histograms generated for cells derived at different times in the developmental sequence. Each point on the curve represents the number of cells having that intensity of fluorescence. A saturation curve was first established using varying concentrations of Fab’. 100% of the cells were stained at the saturating concentration (3.5 mg/ml). The histograms derived for vegetative, 6 hr and 12 hr cells are reasonably monomodal, indicating a homogeneous population of cells expressing gpl50 on their cell surfaces; however, the histogram derived for 18 hr cells is apparently bimodal (peaks roughly at channel numbers 200 and 300), indicating two populations of cells distinguished by their abilities to express gpl50 at the cell surface. It is at this point in development that cells begin to undergo terminal differentiation into
Effect of Fab’ to gp150 on Cell Aggregation If gpl50 were intimately involved in the mechanics of cell aggregation, one would expect that specific coating of the molecule with monovalent Fab’ would abrogate its ability to interact with neighboring cells in a cognitive fashion, thereby resulting in a blockage of cell aggregation. This strategy has been used in a number of systems (Beug, Katz and Gerisch, 1973; Rosen, Chang and Barondes, 1977; Brackenbury et al., 1977). We dissociated 15 hr cells into a single cell suspension, treated them with either Fab’ derived from anti-gpl50 or Fab’ from preimmune animals (or bovine serum albumin) and allowed the cells to reaggregate. The extent of aggregation in these experiments was determined by measuring the absence of single cells as they were being recruited into multicellular aggregates. Figure 6 visually demonstrates these events: at the onset of the incubation, the cells are completely dissociated (A); within 10 min, large multicellular aggregates appear, while the number of single cells is severely reduced (B); when the incubation mixture contains anti-gpl50-Fab’ at a final concentration of 0.6 mg/ml, the cells remain largely dissociated, even up to 1 hr of incubation (C). If the ability of anti-gpl50Fab’ to block cell aggregation is assayed as a function of Fab’ concentration, the curve in Figure 7 is derived. Complete blockage of aggregation essentially occurs at a final anti-gpl50-Fab’ concentration of 0.6 mg/ ml. Three different preimmune Fab’ preparations (including that from the rabbit eventually used as a source of anti-gpl50-Fab’) were tested, and all were found to be without effect on the aggregation process. Specificity of Immune Inhibition of Cell Aggregation In an attempt to delineate further the specificity of the blockage of cell aggregation by anti-gpl50-Fab’, we tested two other antisera with different immunospecificities: one was derived to gpl30. a cell sut-faceassociated glycoprotein whose expression is not under developmental regulation; the other was raised to
Cell 394
Figure 4. Fluorescence Histograms of Cells Taken at Various Times in Development and Treated with Antigpl50-FITC-Fab’ Cells were taken at various points in development, treated with a saturating amount of antigpl50-FITC-Fab’ and analyzed by an FACS II cell sorter as described in Experimental Procedures. Each point on the curve represents a number of cells with a certain intensity. Note that the distributions for 0. 6 and 12 hr cells are monomodal; however, 16 hr cells display a bimodal distribution with respect to gpl50 expression at the cell surface (peaks at roughly channel numbers 200 and 300).
intact vegetative ceils. The specificity of each serum, when tested by immunoprecipitation of total Con A binding proteins, is depicted in Figure 8A. As noted before, anti-gpl50 specifically recognizes gpl50 (lane l), the extraneous bands also being present in the preimmune control (lane 4). Similarly, immunoprecipitation with anti-gpl30 results in a substantial enrichment for gp130 (lane 2). It also appears that the level of gp70 is enhanced in these precipitates, which we occasionally detect in anti-gpl50-derived precipitates. Anti-vegetative cell serum recognizes gp70, gpl30 and gpl50 in these immunoprecipitation analyses (lane 3). The extraneous bands detected in these precipitates, which are also present in the preimmune situation, do not represent specific interaction between antigen and antibody. Preimmune serum does not label the cell surface in either direct or indirect immunofluorescence studies, nor do Fab’s from preimmune sera affect cell aggregation. Since effective blockade of cell aggregation results from the binding of the Fab’ to the cell surface, we first quantitated the binding abilities of the various Fab’s to the surfaces of cells allowed to develop for 16 hr (Experimental Procedures). Cells treated in such a way bound virtually equivalent levels of all three FITC-Fab’s tested (Figure 88). Each Fab’ was then tested for its ability to block cell aggregation. Since all three Fab’s bound with equal efficiency to the cells, a single concentration of Fab’, which was the level that causes complete inhibition of aggregation by anti-gpl50-Fab’, was tested. When the abilities of anti-gpl30-Fab’ and anti-vegetative cell Fab’ were measured relative to anti-gp150 to block cell aggregation, it was found that the former was 1 1 %, the latter 22% as effective as anti-gpl50Fab’ in this regard. It is worth noting that anti-vegetative cell serum does precipitate some gp150; this
OJ Figure 5. Expression Time in Development
,
I
10 15 5 Time in Oevelopement [hrs] of gpl50
on the Cell Surface
20
as a Function
of
Means were calculated from both the fluorescent histograms shown in Figure 4 and histograms derived from light scatter data on the same cells (data not shown). By dividing the scatter mean into the fluorescent mean (ordinate), the binding is expressed as relative fluorescence intensity per unit area.
activity, no doubt, contributes some blockage activity in the cell aggregation assay. These results demonstrate that although all three antisera bind with equal efficiency to the surface of a cell allowed to develop for 16 hr, only the antiserum with specificity to gpl50 is effective in preventing cell aggregation. Discussion Our initial observation that the appearance on the cell surface is a developmentally
of gpl50 regulated
Cell Surface 395
Figure
Glycoprotein
6. Aggregation
in D. discoideum
of 16 Hr Cells in the Presence
of Anti-gpl
BO-Fab’
16 hr cells were harvested, dissociated into single cells and treated with Fab’ as described in Experimental Procedures. (A) Fully dissociated 16 hr cells at the start of the incubation; (B) cells after 10 min of incubation in the presence of 0.6 mg/ml preimmune Fab’; note the large multicellular aggregates and the small number of single cells; (C) cells after 10 min of incubation in the presence of 0.6 mg/ml anti-gpl50-Fab’. Phase optics were utilized. Magnification 100X.
event, and that this expression closely matches the onset of mutual cellular cohesiveness, suggested that this molecule may be intimately involved in the mechanics of cell aggregation of D. discoideum. The data in the present report substantiate this notion; however, a number of caveats must still be considered. It is possible that the interaction between a particular Fab’ and its antigen could result in a selective steric impairment of another set of molecules, those which are authentically involved in the mechanics of cell adhesion. Second, the molecule under study may exert a controlling or modulating influence on other plasma membrane-associated molecules, and perturbation of its function could therefore have pleiotropic consequences. Perhaps a more fundamental concern in these types of experiments is the biological relevance of the cell aggregation events measured in vitro. It is of interest to compare these findings on the involvement of gpl50 in cell aggregation in Dictyostelium with two other systems which have undergone detailed study. A large cell surface-associated proteoglycan complex has been shown to be involved in mediating cell cohesion in the sponge (Henkart, Humphreys and Humphrey& 1973). In this highly specialized case, aggregation of cells apparently occurs as a result of the interaction between an extracellular matrix consisting of this proteoglycan complex and cell surface-associated receptors (Muller et al., 1978). The overall process is species-specific. The second
0
.i
.i
.i
.i
.!I .6 .i
.i
.3
1:o
111
1:2
W’l IWmll Figure 7. Aggregation of 16 Hr Cells Concentrations of Anti-gpl50-Fab’
in the Presence
of Varying
16 hr cells were dissociated and treated with varying amounts of antigpl50-Fab’. Aggregation was allowed to proceed for 30 min. and the number of single cells remaining was determined by the particle counter as described in Experimental Procedures. Similar incubations were set up using Fab’ from a preimmune serum at two different concentrations (0.5 and 2 mg/ml); the number of single cells left after 30 min in both cases was identical and used to generate the zero concentration point on the graph.
system which has undergone extensive investigation is the neural retina cells of the chick embryo. Aggregation between these cells is due, in part, to the action of a cell surface-associated glycoprotein of molecular weight 140,000 daltons (Thiery et al., 1977). Treat-
Cell 396
Immunospecificity
A.
Figure 8. Specificity of Block of Cell Aggregation
Anti-gpl
BO-Fab’
MWws3
(A) lmmunoprecipitation of “‘1 total cellular Con A binding protein. Approximately 1 x 10’ cpm of ‘251-Con A binding protein from 16 hr 150cells were reacted with an appropriate dilution of each antiserum as described in Experimen130tal Procedures. Immune complexes were retrieved with S. aureus; following three washes 70in a-mm in TN, the pellets were boiled in electrophoresis sample buffer and subjected 56 to SDS-PAGE, and an autoradiogram was derived from each lane. (Lane 1) Immunoprecip45' itation with anti-gpl50; 10 ~1 of i/40 dilution of antiserum were used: note that the extraneous bands present here are also present in the preimmune incubations (lane 4). (Lane 2) lmmunoprecipitation with anti-gpl30; 30 pl of l/20 dilution of antigpl30 serum were used to generate this lane; note the enhanced reactivity for gp130 in this case over the other B. Amount of PITC-Fab' Bound at Saturation lanes in the figure. (Lane 3) ImmunoprecipitaRelative Fluorescence tion with anti-vegetative cell; 30 AI of a l/20 Fab' Units dilution were used in this case; when tested anti-gpl50 100 with this antigen (whole cell Con A binding anti-gp130 113 proteins). the antiserum reacts with gpl50. anti-vegetative cell 121 gpl30 and gp70. (Lane 4) Immunoprecipitation with preimmune serum: 10 f.rI of a l/20 C. Ability of Various Fab"s to Block Cell Aggregation dilution of a preimmune serum were used; a number of different preimmune sera gave proNumber of X Inhibitiion Relative files similar to this one. R Pab' Single Cells to Anti-gpl50-Pab' x (B) Saturation binding of FITC-Fab’ to 16 hr cells. Dissociated 16 hr cells were treated with 1.840 100.0 anti-gp150 15,184 6,464 .207 11.3 anti-gp130 the appropriate FITC-Fab’. and the amount of cell 7,755 .440 24.0 anti-vegetative fluorescence bound to the cell was quantitated 5,354 0 0 pre-imune as described in Experimental Procedures. The Fab’s had virtually identical FITC to protein ratios, so that these numbers are directly comparable. These data represent the averages of two separate experiments, where duplicate samples were run for each separate Fab’. (C) Ability of Fab’s of various specificities to block cell aggregation. Cells allowed to develop for 16 hr were dissociated into a single cell suspension and reacted with the appropriate Fab’, each at a fixed concentration of 0.7 mg/ml. Afler 30 min of shaking at 25°C. the extent of aggregation was measured with a particle counter, as outlined in Experimental Procedures. For these experiments, an index of inhibition of aggregation (R.) has been defined: R. =
number
of single cells after incubation number
in X-number
To compare directly the effect of the various the particular Fab’ relative to that of anti-gpl50 Rx R anti-gpl50
of single
of single cells afler incubation
cells after incubation in preimmune
Fab’s on cell aggregation is derived as follows:
relative
in preimmune
Fab’
Fab’ to the effect
by anti-gpl50-Fab’.
a percentage
of inhibition
by
x 100
ment of cells with Fab’ fragments rendered specific to this molecule blocks cell aggregation. The means by which this molecule mediates cell cohesion remains to be elucidated. The relationship between gpl50 and contact site A, which is the molecule(s) ascribed by Gerisch and coworkers to be responsible for cell aggregation in D. discoideum, is unclear. Recently, contact site A has been isolated and shown to consist of a single glycoprotein of approximate molecular weight 80,000 daltons (Huesgen and Gerisch, 1975). Thus contact site A and gpl50 do not appear to be the same molecule; however, the two molecules could still be antigenically related and the difference in size could be due to glycosylation. In an attempt to answer this question,
we derived a preparation enriched in contact site A (Huesgen and Gerisch, 1975). When this material was radioiodinated and immunoprecipitated in the standard fashion with anti-gpl50, we did not detect a band in the 80,000 dalton region of the gel. It therefore appears that gpl50 and contact site A are unrelated molecules. A mass of evidence has been presented which implicates a cell surface-associated carbohydrate binding protein as serving a crucial role in slime mold aggregation (Rosen et al., 1973, 1975; Siu et al., 1978; Ray, Shinnick and Lerner, 1979). The relationship, if any, between gpl50 and this protein remains to be studied. It is certainly possible, and perhaps likely, that cell aggregation is a consequence of a
Cell Surface 397
Glycoprotein
in D. discoideum
number of molecular systems operating at the cell surface, and that contact site A, carbohydrate binding protein and gpl50 belong to different systems. Experimental
Procedures
Cell Growth The wild-type strain NC-4 was used solely in these studies. Conditions for cell growth, development and harvest are identical to those previously described (Geltosky et al., 1976). Preparation of Total Cellular Con A Binding Protein For large-scale preparative purposes, 5 X 10” 16 hr cells were harvested from pads and lysed in 0.5% NP40. 10 mM NaN3 and 0.01% phenylmethylsulfonylfluoride made up in 25 mM Tris. 150 mM NaCl (pH 7.5) (TN). The cells were further disrupted by ultrasonication in a 20KHz Branson sonifer S125 equipped with a one eighth inch probe, and the resulting material was centrifuged at 10,000 rpm for 15 min in a refrigerated Sorvall. The supernatant was then applied to a 5 ml Con A-Sepharose column containing 15 mg of ConA per ml settled beads. The affinity column was prepared as previously described (Geltosky et al., 1976). Following sample application. unbound material was washed off with TN at an approximate flow rate of 0.5 ml/min. When the output absorbance at 280 mp fell to 0.1, specific elution of the glycoproteins by 0.2 M a-methyl-D-mannoside (a-mm) in TN was initiated. The material corresponding to the absorbance peak was pooled, exhaustively dialyzed against HP0 and finally lyophilized. Isolation of gpls0 Approximately 2.5 mg of the above isolated total Con A binding protein was loaded onto a 7% acrylamide gel. To localize gpl50 on the gel without staining for protein, dansylated gammaglobulin was run in a neighboring lane. Conventional protein staining results in fixation of the molecules in the gel, a step we felt wise to avoid, since further loss of native antigenicity and difficulty in recovery of material would accrue. Native gammaglobulin (approximate molecular weight 155,000) has roughly the same electrophoretic mobility as gpl50. Dansylated gammaglobulin was visualized on the gel with the aid of an ultraviolet light: a strip (approximately 2 mm wide) was then excised from the gel corresponding to this location, and gpl50 was eluted from the acrylamide with water in an overnight incubation. This material was then lyophilized and subsequently used for immunization of rabbits. Preparation of Antisera, Gammaglobulin and Fab’ In order to generate antibodies to gpl50, approximately 60 gg of lyophilized gpl50 were mixed with incomplete Freund’s adjuvant and injected subcutaneously into a rabbit. This protocol was repeated fortnightly over a 6 week period. At this time, antibody activity to gpl50 was detected in the serum of the immunized animals. Thereafler. the animals received monthly boosts of gpl50. To obtain gpl30 for immunization purposes, total Con A binding protein was run on 7% SDS gels and gpl30 was visualized by Coomassie brilliant blue staining. A number of bands corresponding to gpl30 were excised from the gel, and the proteins were retrieved by electrophoresis of the isolated slices. Following dialysis and lyophilization, 50-60 pg were injected into a rabbit according to the same protocol as that used for gpl50. Anti-vegetative cell antiserum was generated by injecting approximately 1 X 10’ vegetative cells mixed in incomplete Freund’s adjuvant into a rabbit using the same injection schedule as above. The gammaglobulin fraction of serum was isolated by conventional means: 50% saturated ammonium sulfate precipitation. followed by elution from an anion-exchange resin. Monovalent Fab’ fragments were prepared according to established procedures with some modification (Nisonoff et al., 1960). Divalent F(ab’), fragments were prepared by treating gammaglobulin with pepsin (3% w/w) at pH 4.0 for 4 hr at 37°C. The digest was then dialyzed versus TN and
subjected to chromatography on Sephadex G-l 50 to yield divalent F(ab’), devoid of undigested gammaglobulin and remnants of Fc. Monovalent Fab’ was obtained by first reducing divalent F(ab’), in 0.02 M dithiothreitol for 2 hr at room temperature, followed by alkylation in 0.08 M iodoacetamide for 30 min at room temperature. Following dialysis versus 17 mM phosphate (pH 6.4)(PB). monovalent Fab’ was stored in aliquots at -2O’C. Intact gammaglobulin was not detected in these materials when tested by immunodiffusion or SDSPAGE. When monovalent Fab’ directed to gp150 was tested for antigen binding ability, either by immunoprecipitation or immunofluorescence, strong reactivity was detected. lmmunoprecipitation To test the specificity of the various antisera used in these experiments, immunoprecipitation of radioiodinated total cellular Con A binding protein was utilized. For routine analysis, the following protocol was used. Total Con A binding protein was radioiodinated by chloramine T oxidation to give an approximate specific activity of 3 x 1O5 cpm/pg of protein (McConahey and Dixon, 1966). For immunoprecipitation. the reaction mixture typically contained 8 X 1 O6 cpm of 1251glycoprotein, from lo-20 ~1 of antisera diluted from I:20 to 1: 50, and 100 pl of 0.1 M a-mm, 0.6 M NaCI. 10m4 M KI. and 25 mM Tris (pH 7.5). Mannoside was included in the incubations to negate any interactions between glycoprotein and small amounts of Con A which may have washed off the column during the preparation of the glycoproteins. The incubations were carried out overnight at 4°C; immune complexes were then collected with the addition of 25 ~1 of a suspension of formalin-treated Staphylococcus aureus (Kessler. 1975). Following a 30 min incubation at room temperature, S. aureus was collected by centrifugation and washed 3 times in 0.1 M a-mm, 0.6 M NaCI. 10m4 M KI and 25 mM Tris (pH 7.5). The final pellet was then boiled in electrophoresis sample buffer, the S. aureus was centrifuged out and the supernatant was subjected to SDS-PAGE. Following electrophoresis. the gel was dried down and an autoradiogram was derived through use of Kodak X-Omat RP film. A second method of immunoprecipitation analysis was also used occasionally. Gammaglobulin was prepared from immune serum and coupled to Sepharose to give a final concentration of 5 mg of gammaglobulin coupled per ml of packed Sepharose (Porath et al.. 1973). Approximately 1 x 10’ cpm of ‘251 Con A binding protein were added to 0.5 ml settled volume of anti-gpl50 gammaglobulin Sepharose and 1 ml of 0.1 M a-mm, 2 mg/ml bovine serum albumin in TN. This mixture sat at 37°C for 2 hr with occasional mixing. Following this incubation period, the beads were collected by centrifugation and washed 3 times in the above buffer. The final pellet of beads was treated with electrophoresis sample buffer for 1 hr at 37’C. Following this, the beads were centrifuged and the supernatant was subjected to SDS-PAGE. Electrophoresis Conditions Electrophoresis of proteins was carried out as previously described (Geltosky et al., 1976). 7% acrylamide gels were solely used. Direct lmmunofluorescence To carry out direct immunofluorescence of cells for the cell sorter experiments, the Fab’ was labeled with fluorescein isothiocyanate (FIT0 to yield 1.6 molecule of FITC per molecule of Fab’ (Clark and Sheppard, 1963). Approximately 2 X lo6 cells were labeled with FITC-Fab’ at 5 mg/ml in 20 mM EDTA in PB (DS) in a volume of 50 ~1 for 20 min at room temperature. The cells were washed 6 times in cold DS and kept on ice prior to cell sorter analysis. In order to obtain a quantitative estimate of the amount of fluoresceinated antibody bound to the cells, labeling was carried out as above. At the end of the washing procedure, the cells were lysed in 0.6 ml of 0.1 N NaOH. Fluorescence was measured in a Perkin-Elmer Model MPF-44A. using an excitation wavelength of 490 rnp and an emission wavelength of 530 mp. To insure linearity of response and also to derive the number of FITC-Fab’ molecules bound to the cell surface, a standard curve was constructed by adding known amounts of FITC-Fab’ to a cell extract prepared in 0.1 N NaOH.
Cdl 398
Flow Cytofluorimetry Cells labeled with FITC-conjugated antibody were analyzed by light scatter (to give cell size) and fluorescence on a Becton-Dickinson FACS II fluorescence-activated cell sorter. The laser was operated at 400 mW and the photomultiplier tube at 600 V. 5 c fluorescent beads (Coulter) were used to calibrate the machine.
Kessler, S. (1975). Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameter of the interaction of antibody antigen complexes with protein A. J. Immunol. 115. 1617-1624.
Cell Aggregation Assay The ability of the various monovalent Fab’s to block reaggregation of aggregation-competent cells was assayed by measuring the decrease in the number of single cells remaining after a given time of incubation. Cells allowed to develop for 16 hr were dissociated into a single cell suspension by repeated pipettings in DS. The cell concentration was adjusted to 1 x 1 07/ml and 50 ~1 were added to a 12 x 75 mm glass culture tube. An equal volume of Fab’ solution (made up in PB) was then added, and the tubes were allowed to sit on ice for 30 min. At the end of this time, the tubes were vortexed and then shaken at 150 rpm on a New Brunswick shaker (model Gl 0) maintained at 22’C. The aggregation reaction was terminated upon pipetting the entire reaction volume into 20 ml of 10 mM EDTA in PB. In general, triplicates were run for each determination. Single cells were counted in a Coulter Particle Counter (model 261) using the following settings: 1 /amplification = l/2, 1 /aperture current = 1, lower threshold = 10 and upper threshold = 60. Adherence to these conditions allowed 90% of the single cells to be counted. For each sample, two readings were made, each expending 0.5 ml of the total 20 ml. As a control for each experiment, incubations were carried out in the presence of either Fab’ from a preimmune rabbit or bovine serum albumin. Identical results were achieved with both these controls.
Mtiller. K. and Gerisch. G. (1978). A specific glycoprotein as the target site of adhesion blocking Fab in aggregating Dictyostelium cells. Nature 274, 445-449.
Acknowledgments We wish to thank Dr. Juan Yguerabide and Dr. Al Jesaitis of UCSD for providing us with the spectrofluorometer and advice concerning the fluorescence experiments. This research was supported in part by a research fellowship from the American Cancer Society. This is publication number 106 from the Department of Cellular and Developmental Immunology. and number 1763 from the Research Institute of Scripps Clinic, La Jolla, California. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
May
15, 1979;
revised
Beug. H.. Katz, F. E. and Gerisch, G. (1973). Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum. J. Cell Biol. 56, 647-658. Brackenbury. R.. Thiery. J.-P., Rutishauser. U. and Edelman, G. M. (1977). Adhesion among neural cells of the chick embryo. J. Biol. Chem. 252, 6835-6840.
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Ray, J., Shinnick. T. and Lerner. R. A. (1979). A mutation altering the function of a carbohydrate binding protein blocks cell-cell cohesion in developing Dictyostelium discoideum. Nature 279, 215-221, Rosen, S. D.. Chang, C.-M. and Barondes. S. H. (1977). Intercellular adhesion in the cellular slime mold Polyspondilium pallidurn inhibited by the interaction of asialofetuin or specific univalent antibody with endogenous cell surface lectin. Dev. Biol. 61, 202-213. Rosen, S. D.. Reitherman, R. W. and Barondes, S. H. (1975). Distinct lectin activities from six species of cellular slime molds. Exp. Cell Res. 95, 159-l 86. Rosen, S. D.. Kafka, J. A., Simpson, D. L. and Barondes, S. H. (1973). Developmentally regulated carbohydrate binding protein in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA 70. 25542558. Siu. C-H.. Lerner, R. A.. Ma, G.. Firtel. R. A. and Loomis, W. F. (1976). Developmentally regulated proteins of the plasma membrane of Dictyostelium discoideum. The carbohydrate-binding protein. J. Mol. Biol. 100, 157-l 78. Thiery, J.-P., Brackenbury. R.. Rutishauser. U. and Edelman, G. M. (I 977). Adhesion among neural cells of the chick embryo. II. Purification and characterization of a cell adhesion molecule from neural retina. J. Biol. Chem. 252, 6841-8845.
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