The cadA gene in Dictyostelium encodes the Ca2+-dependent cell adhesion molecule DdCAD-1, which is expressed soon after the initiation of development. To investigate the biological role of DdCAD-1, the cadA gene was disrupted by homologous recombination. The cadA-null cells showed a 50% reduction in EDTA-sensitive cell adhesion. The remaining EDTA-sensitive adhesion sites were resistant to dissociation by anti-DdCAD-1 antibody, suggesting that they were distinct adhesion sites. Cells that lacked DdCAD-1 were able to complete development and form fruiting bodies. However, they displayed abnormal slug morphology and culmination was delayed by ∼6 hours. The yield of spores was reduced by ∼50%. The proportion of prestalk cells in cadA slugs showed a 2.5-fold increase over the parental strain. When cadA cells were transfected with pcotB::GFP to label prespore cells, aberrant cell-sorting patterns in slugs became apparent. When mutant prestalk cells were mixed with wild-type prespore cells, mutant prestalk cells were unable to return to the anterior position of chimeric slugs, suggesting defects in the sorting mechanism. The wild-type phenotype was restored when cadA cells were transfected with a cadA-expression vector. These results indicate that, in addition to cell-cell adhesion, DdCAD-1 plays a role in cell type proportioning and pattern formation.

Cell-cell interactions mediated by adhesion molecules underlie many key events during development, such as cell sorting and cell-type proportioning, which are the bases of pattern formation. Dysfunction of cell adhesion molecules often leads to diseases and abnormalities in fetal development. The social amoeba Dictyostelium discoideum offers many useful features as a model system for the study of cell-cell interactions during morphogenesis. It possesses a simple and well-defined life cycle (for a review, see Loomis, 1975). Upon starvation, cells embark on a developmental cycle leading to the formation of a fruiting body. At the early stages of development, cells migrate in response to extracellular cAMP to form large aggregates, which eventually develop into pseudoplasmodia, also known as slugs. At the cell mound stage, cells begin to differentiate into several major cell types, which sort out from each other to give rise to a spatial pattern with prestalk cells in the anterior quarter of the slug and prespore cells in the posterior region. In addition, a small number of anterior-like cells are scattered among the prespore cells. During culmination, each slug will form a fruiting body consisting of a sorus of spores supported on top of a filamentous stalk and a basal disc.

During development, Dictyostelium cells express several adhesion systems that allow cells to adhere to each other as they aggregate (for reviews, see Fontana, 1995; Siu et al., 1997). Early studies by Gerisch distinguished two major classes of cell adhesion sites (Gerisch, 1980). One class is sensitive to low concentrations of EDTA, while the other is stable in EDTA up to a concentration of 15 mM (Beug et al., 1973). The EDTA-sensitive cell adhesion sites can be divided into two subtypes, the EDTA/EGTA-sensitive adhesion sites and the EDTA-sensitive/EGTA-resistant adhesion sites (Fontana, 1993). The EDTA/EGTA-sensitive sites are mediated by the cell adhesion molecule DdCAD-1/gp24, which is encoded by the cadA gene and appear soon after the initiation of development (Knecht et al., 1987; Brar and Siu, 1993; Yang et al., 1997). The EDTA-sensitive/EGTA-resistant sites appear at 2 hours of development and they are probably dependent on Mg2+ (Fontana, 1993). Expression of the Ca2+/Mg2+-independent cell adhesion molecule gp80 is induced at the onset of cell aggregation, and becomes maximal at the mid-aggregation stage (Murray et al., 1983; Siu et al., 1985). gp80 molecules preferentially associate with raft-like domains in the plasma membrane (Harris et al., 2001a; Harris et al., 2001b) and they mediate cell-cell adhesion via a homophilic binding mechanism (Siu et al., 1987; Kamboj et al., 1988; Kamboj et al., 1989; Stein and Gerisch, 1996). Upon the formation of loose aggregates, cells express another Ca2+/Mg2+-independent cell adhesion molecule, gp150, which mediates cell-cell adhesion by heterophilic binding and is likely to be involved in the sorting out of prespore cells and prestalk cells (Siu et al., 1983; Gao et al., 1992; Wang et al., 2000).

DdCAD-1 is a unique cell adhesion molecule because it does not contain a signal peptide or a transmembrane domain (Wong et al., 1996). It is synthesized as a soluble protein in the cytoplasm and then transported to the plasma membrane by contractile vacuoles (Sesaki et al., 1997). DdCAD-1 molecules on the cell surface can be induced to form ‘caps’ by antibody crosslinking, suggesting that they are linked to the cytoskeleton by a transmembrane component. DdCAD-1 shows limited sequence similarities with classical cadherins (Wong et al., 1996). Similar to cadherins, DdCAD-1 is a Ca2+-binding protein and its adhesive activity is dependent on Ca2+ (Brar et al., 1993; Wong et al., 1996). DdCAD-1 is found concentrated on filopodia and in contact regions between apposing cells (Sesaki and Siu, 1996). Moreover, high levels of secreted DdCAD-1 in the medium have an anti-adhesion effect (Siu et al., 1997).

To investigate the biological roles of DdCAD-1 during Dictyostelium development, cadA mutants were generated by gene disruption. The EDTA/EGTA-sensitive cell adhesion was abrogated in cadA cells. Many slugs displayed abnormal morphology and the culmination stage was delayed. Although mutant cells were able to complete development and formed fruiting bodies, defects in cell-type proportioning and cell sorting were observed.

Cell strains and culture conditions

The Dictyostelium axenic strain KAX3 was cultured in HL-5 medium and used in all transformation studies. Subsequently, KAX3 and the transformants were cultivated at 22°C, either axenically in HL-5 medium or on SM agar plates in association with Klebsiella aerogenes (Sussman, 1987). For development, cells were either plated on 2% non-nutrient agar or shaken at 180 rpm in 17 mM phosphate buffer, pH 6.4, at 2×108 cells/ml. To observe slugs, cells were developed on agar in the dark to delay fruiting body formation.

Construction of integration vector and cell transformation

The integration vector pbsrΔBgl, with the BglII site disrupted (Adachi et al., 1994) was used for the construction of the transformation vector. The blasticidin S-resistance (bsr) gene is included in the integration vector as a selectable marker in Dictyostelium transformants (Sutoh, 1993). In constructing the plasmid for the disruption of cadA, the EcoRI site of pbsrΔBgl was eliminated by blunt-end ligation. The resulting plasmid was named pbsrΔEco. A 3.8 kb EcoRI fragment containing the cadA gene (GenBank Accession Number, AF340153) was isolated from a genomic λgt10 library (C. Y. and C.-H. S., unpublished). This 3.8 kb DNA fragment was cut out from the plasmid with EcoRI and circularized and then cut at the HincII site within the cadA gene. The linear plasmid DNA was then subcloned into the blunt-ended SmaI site of pbsrΔEco to generate pbsr/cadA (Fig. 1A,B).

KAX3 cells were cultured axenically in HL-5 medium to 2-4×106 cells/ml. After chilling on ice for 15 minutes, cells were washed once with cold electroporation buffer, which contained 10 mM phosphate buffer (pH 6.4) and 50 mM sucrose (Adachi et al., 1994), and then resuspended in the same buffer at 107 cells/ml. The cell suspension was added to 10 μg of pbsr/cadA linearized with EcoRI and 25 units of EcoRI, to give a final volume of 0.8 ml. The plasmid DNA was introduced into cells by electroporation using the BioRad Gene Pulser with settings of 0.9 kV and 3 μF according to the method of Kuspa and Loomis (Kuspa and Loomis, 1992). The time constant ranged from 0.9 to 1.1 mseconds. After 15 minutes on ice, the cell suspension was mixed with 8 μl of 100 mM MgCl2 and 100 mM CaCl2. Aliquots (200 μl) of cells were placed on 100 mm plastic culture dishes. To each dish, 10 ml of HL-5 medium was added and cells were dispersed gently. After 15 hours of incubation at 22°C, blasticidin S (Sigma) was added to a final concentration of 10 μg/ml. The transformants were grown at 22°C without a medium change. Visible colonies appeared within 3 days. Transformants were picked and assayed for loss of DdCAD-1 expression. Clones with no detectable DdCAD-1 were recloned once and then subjected to further characterization.

DNA isolation and Southern blot analysis

Genomic DNA was prepared from 108 KAX3 cells, which were lysed in 20% SDS. The cell lysate was incubated for 10 minutes at 65°C and then extracted with phenol and chloroform. The aqueous phase was treated with RNase A (1 mg/ml) and proteinase K (2 mg/ml) for 1 hour at 37°C, followed by phenol-chloroform extraction before the DNA was precipitated with cold ethanol.

Genomic DNA was cut using different restriction enzymes and the fragments were separated on an agarose gel and then transferred to a nitrocellulose membrane. DNA hybridization was carried out using 32P-labeled cadA cDNA and pbsrΔEco DNA as probes for 18-20 hours at 42°C in 50% formamide and 5×SSC (20×SSC, 3 M NaCl and 0.3 M sodium citrate). The filters were washed for 30 minutes each, first at room temperature with 2×SSC, and then under more stringent conditions at 65°C in 2×SSC plus 1% SDS, and finally in 0.1×SSC plus 0.1% SDS.

Developmental morphology of mutant strains

Mutant cells were cultured on agar plates in association with Klebsiella aerogenes (Sussman, 1987). To examine synchronous morphological development, cells were washed free of bacteria and then resuspended in 17 mM phosphate buffer, pH 6.4, for development on 2% non-nutrient agar at ∼106 cells/cm2. To quantify the yield of spores, cells were developed on 2% plain agar plates for 36 hours. Fruiting bodies were collected from the agar plates and then centrifuged. The pellet was frozen and thawed once before suspending in 1 ml of 17 mM phosphate buffer, pH 6.4, containing 0.1% SDS. The number of spores was counted using a hemocytometer. Spore viability was tested by plating the spores on SM-agar plates in association with bacteria, and colonies were counted 3 days later.

Cell cohesion assay

Cell cohesion assays were performed using a modified method (Lam et al., 1981) of the original roller tube assay of Gerisch (Gerisch, 1961). Cells were collected for development in 17 mM phosphate buffer (pH 6.4) at 2×107 cells/ml. After 4 hours, cells were resuspended at ∼2.5×106 cells/ml. Cell aggregates in 200 μl were dispersed by vortexing for 15 seconds. Cells were allowed to re-form aggregates on a platform shaker rotating at 180 rpm at room temperature. At regular time intervals, the number of non-aggregated cells, including singlets and doublets, were scored using a hemocytometer. The percentage of cell aggregation was calculated by dividing the difference of the total number of cells and the number of singlets and doublets by the total number of cells. To assess the requirement for divalent cations, cells were assayed in the presence of 10 mM of EDTA or EGTA. The inhibitory effect of carnitine, which inhibits the EDTA-sensitive cell adhesion site (Desbarats et al., 1994), was also examined. Inhibition studies on the EDTA-resistant adhesion sites were carried out using the murine mAb 80L5C4, which recognizes the homophilic binding site of gp80 (Siu et al., 1985; Wu et al., 1992).

Staining of prestalk cells with Neutral Red

Wild-type and mutant strains were cultured in liquid medium. Cells were washed twice in 17 mM phosphate buffer (pH 6.4) and resuspended at 107 cells/ml. The cells were stained by incubation in phosphate buffer containing 0.03% Neutral Red for 5 minutes at room temperature (Weijer et al., 1987). After washing, cells were deposited on 2% non-nutrient agar at 5×105 cells/cm2. Development was carried out in the dark at 22°C for different times. Single slugs were picked up using a plastic pipette tip and transferred to a microfuge tube containing 20 mM EDTA in 17 mM phosphate buffer. Cells were washed twice with buffer and the percentage of Neutral Red-stained cells was determined.

Cell sorting in mixed aggregates

Cells were developed for 4 hours and then incubated with DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) at 12.5 μg/ml. DiI-labeled cells were mixed with unlabeled cells at a ratio of 1:1 in Bonner’s salt solution containing 10 mM NaCl, 10 mM KCl and 3 mM CaCl2. Cells were resuspended at 5×107 cells/ml and placed in 24-well tissue culture plates. Cell samples were rotated at 150 rpm for 6 hours to allow cell sorting to take place in aggregates. Approximately 100 aggregates were scored visually for each assay. Aggregates showing clear segregation of the labeled cells from the unlabeled cells were scored as ‘sorted’, while the ‘unsorted’ ones had labeled cells interspersed among the unlabeled cells in the aggregate.

Cell sorting in chimeric slugs

To construct chimeric slugs, the anterior one-quarter was dissected from Neutral Red-stained cadA slugs that showed a ‘sorted’ pattern, while posterior half was dissected from unlabeled wild-type KAX3 slugs. Cells were dissociated separately in 20 mM EDTA and then were mixed at a ratio of 1:4 (cadA:KAX3) in Bonner’s salt solution and allowed to re-form slugs on 2% plain agar plates. The distribution pattern of stained cells within the chimeric slugs was examined by light microscopy. Approximately 50 slugs were analyzed in each experiment. The lengths of the whole slug (y) and the anterior Neutral Red-stained zone (x) were measured. The x/y ratio (R) was calculated for each slug and the frequencies of occurrence for these ratios were determined.

Analysis of cells transfected with pcotB::GFP

KAX3 and cadA mutant cells were transformed with a plasmid carrying the construct cotB::GFP and the neomycin-resistant gene cassette for selection, yielding the cell lines, JS24 (cadA+) and TL144 (cadA), respectively. These cells were cultured in HL-5 medium supplemented with 20 μg/ml of G418. As cotB is a prespore-specific gene (Fosnaugh and Loomis, 1993), the promoter drives the expression of GFP in prespore cells of the transformants. The pattern of prespore cell and prestalk cell distribution in slugs was recorded and the lengths of the slug (y) and the anterior zone (x) were measured. When the x/y ratio was <0.3, the slug was considered to have a normal sorting pattern. When a clearly demarcated anterior zone was not observed, the slug was taken to have a non-sorted pattern.

Ectopic expression of DdCAD-1 in cadA cells

Full-length cadA cDNA was prepared by PCR using the forward primer 5′-GGACTAGTATGGTAGTTTGACCTTGT-3′ and the reverse primer 5′-GGCTCGAGATTATTTCTGAAATTCAT-3′ and then cloned into the SpeI and XhoI sites of the actin-15 expression vector of EXP4(+) (Dynes et al., 1994). The plasmid DNA was introduced into cadA cells (cadA-12) by electroporation. Cells were selected in the presence of G418. Stable transformants were maintained at 10 μg/ml of G418 in HL-5 medium.

Disruption of the cadA gene

We have previously shown that cadA is a single-copy gene localized within a 4 kb EcoRI restriction fragment of the Dictyostelium genomic DNA (Wong et al., 1996). In order to isolate the cadA gene, a small λgt10 library was made using EcoRI fragments between 3 and 5 kb, and then probed with the cadA cDNA. A 3.8 kb EcoRI fragment was obtained from several independently isolated clones and two of them were subjected to DNA sequencing. These fragments contained the entire coding sequences of the cadA gene and 2.5 kb of 5′ flanking DNA. The coding region of the cadA gene consists of three exons separated by two short introns (Fig. 1A).

We used this 3.8 kb DNA fragment to construct the integration plasmid. The cadA gene was disrupted by the restriction enzyme-mediated integration method as shown in Fig. 1B (Kuspa and Loomis, 1992; Sutoh, 1993). The blasticidin S-resistance (bsr) gene served as the selection marker in the integration vector pbsrΔEco (Adachi et al., 1994). In one experiment, 31 blasticidin S-resistant transformants were obtained and screened for DdCAD-1 expression. Two independent drug-resistant clones, cadA-10 and cadA-12, did not express DdCAD-1 (Fig. 1C). DNA blot analysis confirmed that the cadA gene was disrupted in these two clones. The cadA cDNA and the integration vector pbsrΔEco were used as probes and they both hybridized with a restriction fragment of ∼9 kb in cadA-10 and cadA-12 cells, consistent with the expected size of a cadA DNA fragment containing the bsr gene cassette (Fig. 1D). However, the cDNA probe, but not the vector probe, hybridized with a 4 kb band of wild-type DNA. In a separate experiment, two independent cadA clones, TL97 and TL98, were obtained. All four clones yielded similar results in subsequent studies and representative data are presented for either cadA-10 or cadA-12.

Cohesive properties of cadA cells

Cell cohesion assays were performed to assess the effects of the loss of DdCAD-1 expression on EDTA-sensitive cell-cell adhesion. Both KAX3 and cadA cells were developed for 4 hours, dissociated mechanically into single cells. Cell reassociation was carried out in the presence of 10 mM EDTA or EGTA. In the absence of chelators, cadA cells showed a 50% reduction in the level of cell reassociation in comparison with KAX3 (Fig. 2). As gp80 was not yet expressed at this time, only EDTA-sensitive cell adhesion sites were present. Indeed, cell reassociation of both KAX3 cells and cadA cells was completely inhibited by EDTA (Fig. 2B). Carnitine, which was found to inhibit cell cohesion in the early phase of development, also inhibited the EDTA-sensitive sites in the cadA cells (Fig. 2C). The addition of EGTA in the assay inhibited KAX3 cell reassociation by ∼50%. However, EGTA failed to exert any effect on cadA cells (Fig. 2B). Furthermore, when mutant cells were reassociated in the presence of either soluble DdCAD-1 or anti-DdCAD-1 Fab, both of which are known to inhibit the Ca2+-dependent cell adhesion sites (Brar and Siu, 1993), neither reagent had an appreciable effect on these EGTA-resistant adhesion sites (Fig. 2C). These results thus indicate that the cadA cells have lost the ability to express the Ca2+-dependent cell adhesion sites and that the EDTA-sensitive/EGTA-resistant adhesion sites are distinct from those mediated by DdCAD-1.

Developmental morphology of cadA cells

The growth rate of cadA mutants in axenic medium was very similar to that of wild-type cells, with a doubling time of ∼12 hours. However, when cells were developed on non-nutrient agar, differences in developmental time and morphology were observed. Mutant cells underwent normal chemotaxis and formed cell streams that resembled parental cells. However, cell mounds appeared 1 to 2 hours earlier than wild-type cells and mound sizes were often larger. The aggregation stage was followed by an extended slug stage, such that culmination was delayed by ∼6 hours. Nevertheless, cadA cells were able to complete the developmental cycle and formed fruiting bodies with viable spores.

Upon close examination, aberrant morphological structures were prevalent between the mound and early slug migration stages (Fig. 3). Aggregates of cadA cells often showed many small nodule-like structures protruding from the surface of the mound (Fig. 3D). Whereas KAX3 aggregates typically develop several tips and split to form slugs of similar sizes (Fig. 3A,B), cadA cell mounds frequently did not split up. If they did, splitting was in an uneven manner and gave rise to slugs of variable sizes. Many of these nodule-like protrusions remained on the finger structures and early migrating slugs (Fig. 3E) and the cadA slugs often appeared longer and more slender than wild-type slugs. Most of the nodule-like protrusions disappeared with slug migration. In comparison with its parental strain, the cadA fruiting bodies often had longer stalks, bigger basal discs and proportionally smaller sori (Fig. 3f). About 25% of the cadA fruiting bodies showed some abnormal morphology. Multiple sori on a single stalk and kinky stalk-like structures protruding from the top of the sori were observed. Only ∼5% of wild-type fruiting bodies had these abnormal features.

Defects in cell-type proportioning in cadA slugs

The above observations suggest that cadA cells might have defects in spore cell differentiation. To monitor spore formation, equal numbers of wild-type cells and cadA cells were deposited on plain agar for development and the fruiting bodies were collected to estimate the total number of spores. The yield of spores was reduced by 40-50% in cadA cells in comparison with wild-type cells (Fig. 4A). When the same amount of KAX3 and cadA spores were plated on agar plates, similar numbers of colonies were obtained, indicating equal viability.

The reduction in spore yield in cadA strains suggested defects in cell-type proportioning in the slug stage, when prestalk and prespore cells sort out from each other to give rise to an anteroposterior pattern. To monitor cell-type differentiation in slugs, cells were labeled with the vital dye Neutral Red, which stains acidic lysosomal vesicles in prestalk and anterior-like cells (Devine and Loomis, 1985; Weijer et al., 1987). The proportion of Neutral Red-stained cells in wild-type and mutant strains was determined at different times between the mound stage (∼10 hours of development) and the late slug stage (∼18 hours). More than 90% of both KAX3 and cadA-10 cells were stained red in the early mound stage. The percentage of stained KAX3 cells decreased at 13 to 14 hours of development when cell-type differentiation occurred and slugs were formed (Fig. 4B). The percentage of Neutral Red-stained cells decreased to ∼25% in KAX3 slugs, closely corresponding to the percentage of prestalk cells normally found in wild-type slugs. By contrast, cadA slugs had only a small drop in the percentage of Neutral Red-positive cells and >60% of cells were stained red between 14 and 18 hours. The increase in Neutral Red-stained cells in cadA slugs suggested an increase in prestalk cells, which might account for the reduction in spores in fruiting bodies.

Aberrant patterns of prestalk and prespore cell distribution in cadA slugs

In wild-type slugs, the Neutral Red-stained cells sorted to the anterior one-quarter of the slug length. By contrast, a variety of abnormal patterns were observed with the cadA slugs (Fig. 5A-C). In addition to the anterior zone, large clusters of stained cells were present throughout the posterior zone of mutant slugs, suggesting defects in cell sorting.

To further examine the prestalk and prespore pattern in slugs, both wild-type and cadA cells were transfected with the expression vector containing GFP cDNA driven by the cotB promoter. As cotB is a strong promoter in prespore cells (Fosnaugh and Loomis, 1993), prespore cells in the slug of transformants would be labeled by GFP. A low level of GFP expression was first observed at the mound stage. When the mutant transformant TL144 (cotB::GFP, cadA) was examined, GFP-expressing cells were present randomly throughout the mound and in the nodule-like protrusions (Fig. 5E), suggesting that GFP-expressing cells and non-expressing cells had not sorted out at this stage. In slugs derived from the wild-type transformant JS24 (cotB::GFP, cadA+), fluorescent prespore cells occupied the posterior three quarters to four-fifths of the slug (Fig. 5D). Quantitative analysis showed that ∼80% of JS24 slugs displayed this ‘sorted’ pattern by 18 hours of development (Fig. 6). By contrast, only 25% of TL144 slugs showed the normal sorting pattern. When slugs without a clearly demarcated anterior zone (Fig. 5F) were scored as ‘non-sorted’, time-course analysis showed that 30% of TL144 slugs displayed a non-sorted pattern at 14 hours compared with <5% for JS24 slugs (data not shown). By 18 hours, the percentages of non-sorted slugs were comparable for both strains. However, the majority of cadA slugs still displayed a variety of aberrant sorting patterns (Fig. 5H). In some slugs, even though a clearly demarcated anterior zone was observed, it often occupied more than one-third of the slug length and non-fluorescent patches of cells were present in the posterior region (Fig. 5G). When these slugs were scored as ‘partially sorted’, they constituted ∼70% of the cadA slug population at 18 hours (Fig. 6).

The increase in regions occupied by non-fluorescent cells suggests an increase in the proportion of non-GFP-expressing cells or prestalk cells in the cadA slugs. Consistent with the data obtained with Neutral Red-stained cells, FACS analysis showed that the cadA slugs contained an average of 50% GFP-expressing cells, while KAX3 slugs had ∼80% GFP-expressing cells (data not shown).

Defects in cell sorting exhibited by cadA prestalk cells

The above results suggested that cadA cells might not be able to undergo proper cell sorting during slug formation. To determine whether cadA cells were defective in cell sorting, we tested whether the Neutral Red-stained cadA prestalk cells in the anterior region of the slug were able to sort out from wild-type prespore cells in chimeric slugs. The Neutral Red-stained anterior zones of cadA slugs with a normal sorted pattern were dissected and mixed with wild-type prespore cells at a ratio of 1:4. The cell mixture was allowed to re-form slugs on an agar surface. The chimeric slugs were examined by epifluorescence microscopy. The lengths of both slugs and anterior regions that contained stained cells were measured and the ratio (R) of these two values was plotted against the frequency of occurrence. As a control, wild-type prestalk cells were mixed with wild-type prespore cells. In this case, the majority of the re-constituted slugs displayed the normal sorting pattern and 80% of the slugs had a R value of <0.4 (Fig. 7). By contrast, the histogram for chimeric slugs shifted to the higher R values, with 55% of the slugs having a R value of >0.4, suggesting that the cadA prestalk cells were inefficient in sorting out from the prespore cells to re-occupy their anterior position.

Altered cell cohesiveness due to precocious gp80 expression in cadA cells

Differential cell adhesiveness is a major driving force in the sorting out process of two randomly mixed cell populations (Steinberg, 1975; Nicol et al., 1999). The aberrant sorting pattern observed in slugs of cadA cells might be due to changes in the profile of cell adhesion molecules. Changes in the adhesive properties of null cells were examined using the cell sorting assay. cadA cells were co-aggregated with wild-type cells to allow the formation of randomly mixed aggregates. In each assay, one cell population was labeled with the fluorescent dye DiI and mixed at 1:1 ratio with the unlabeled cells. Cell mixtures were rotated on a platform shaker for another 6 hours and the percentages of ‘sorted’ aggregates were scored. When labeled KAX3 cells were mixed with unlabeled KAX3 cells or labeled cadA-10 cells with unlabeled cadA-10 cells, 90% of the aggregates displayed a non-sorted morphology (Fig. 8). Therefore, labeling cells with DiI did not affect the adhesive properties of these cells. However, when KAX3 cells were mixed with DiI-labeled cadA cells, almost 80% of the aggregates had a ‘sorted’ morphology (Fig. 8). Although the loss of DdCAD-1 expression in null cells was expected to reduce intercellular cohesiveness, the cadA cells showed a tendency to sort to the center or the sides of most aggregates, suggesting that they were more cohesive than KAX3 cells.

The above results were unexpected. A possible reason might be that the loss of DdCAD-1 expression had an effect on the expression of other cell adhesion molecules. Therefore, we examined the expression of the EDTA-resistant cell adhesion molecule gp80, which normally accumulates on the cell surface during the cell aggregation stage of development. Protein blot analysis showed that gp80 was expressed precociously in cadA cells (Fig. 9A). Moreover, cadA cells accumulated a higher level of gp80 than did wild-type cells. When subjected to the cell cohesion assay, cadA cells acquired Ca2+/Mg2+-independent cell adhesion ∼2 hours earlier and achieved a higher level of cell cohesion than wild-type cells at 10 hours (Fig. 9B). In both mutant and wild-type strains, the EDTA-resistant cell adhesion sites were inhibited by the anti-gp80 mAb 80L5C4.

Ectopic expression of DdCAD-1 restored the wild-type phenotype

To determine whether ectopically expressed DdCAD-1 was able to rescue the cadA-null phenotype, cadA cDNA was introduced into cadA-12 cells. Two clones, WT33 and WT94, which expressed a similar level of DdCAD-1 as did wild-type cells, were chosen for further analysis (Fig. 10A). The cell cohesion assay clearly demonstrated that Ca2+-dependent cell-cell adhesion was restored in the transformants to a level similar to that of KAX3 cells (Fig. 10B). The normal pattern of gp80 accumulation during development was restored (Fig. 10C) and the expression of EDTA-resistant cell adhesion sites followed closely that of KAX3 cells (Fig. 10D).

Furthermore, slugs and fruiting bodies derived from the transformants displayed the wild-type morphology. More than 80% of the slugs exhibited the normal prestalk-prespore segregation pattern. The percentage of prestalk cells in slugs as indicated by Neutral Red staining was reduced to the wild-type level of 25% (Fig. 11A). The spore yield was also comparable to that of wild-type cells (Fig. 11B). Thus, ectopically expressed DdCAD-1 was able to restore the normal developmental phenotype in cadA cells.

In this paper, we have provided genetic evidence that DdCAD-1 mediates Ca2+-dependent cell-cell adhesion among Dictyostelium cells. In addition, disruption of the cadA gene results in abnormal pattern formation in slugs and a reduction in spore yield, implicating a role for DdCAD-1 in cell sorting and cell-type proportioning during Dictyostelium development.

DdCAD-1 was first implicated in cell-cell cohesion based on antibody inhibition studies (Knecht et al., 1987). Subsequently, DdCAD-1 purified from cells, as well as different recombinant DdCAD-1 fragments, have been shown to contain Ca2+-binding and cell-binding activities (Brar et al., 1993; Wong et al., 1996). Here, the function of DdCAD-1 is further borne out by the fact that the inactivation of the cadA gene leads to the loss of Ca2+-dependent cell-cell adhesion during Dictyostelium development (Fig. 2). Re-expression of DdCAD-1 in cadA cells restores the Ca2+-dependent adhesion sites (Fig. 10). However, the cadA cells show only a 50% reduction in EDTA-sensitive adhesion. The remaining adhesion sites are resistant to EGTA, suggesting that they may require Mg2+ and not Ca2+ for their function. Thus, the loss of DdCAD-1 expression has highlighted the presence of a distinct class of EDTA-sensitive cell adhesion sites. Although these sites were reported earlier (Fontana, 1993), their molecular identity is not yet known. The cadA cells should provide a useful model for the future analysis of these adhesion sites.

In addition to cell-cell adhesion, there is growing evidence that cell adhesion molecules are important morphoregulatory molecules and signaling molecules that regulate cell behavior, cell differentiation and other important biological processes (Edelman and Crossin, 1991; Gumbiner, 1996; Hynes, 1999). Indeed, inactivation of the cadA gene not only results in the loss of the Ca2+-dependent cell-cell adhesion, but also gives rise to aberrant morphogenesis. In the mound stage of wild-type cells, prestalk cells sort out from prespore cells and move to the tip, occupying the anterior quarter of the slug. Studies from several laboratories have suggested that cell sorting involves differential cell adhesiveness, differential chemotaxis, relative cell motility or a combination of these processes (Siu et al., 1983; Early et al., 1995; Siegert and Weijer, 1995; Sukumaran et al., 1998; Kellerman and McNally, 1999; Nicol et al., 1999; Clow et al., 2000). Mathematical simulations suggest that the formation of a tip containing only prestalk cells occurs within a narrow range of combined inputs from differential adhesion and chemotaxis (Jiang et al., 1998). While prespore cells are more cohesive than prestalk cells (Lam et al., 1981), prestalk cells are more motile in response to cAMP than prespore cells (Early et al., 1995). These differences may account for the sorting of anterior cells to the peripheral regions at the mound stage.

As DdCAD-1 is excluded from cell-cell contacts in the post-aggregation stages and becomes localized primarily in the cytoplasm (Sesaki and Siu, 1996; Harris et al., 2001b), it cannot directly account for the differential cell adhesiveness between prespore and prestalk cells. Antibody inhibition studies have implicated the cell adhesion molecule gp150 in cell sorting (Siu et al., 1983). In addition to cell adhesion molecules, cell sorting may involve other regulatory components. The disruption of the dtfA gene, which encodes a membrane protein that contains mucin-like motifs, has been reported to modulate both cell-cell adhesion and cell sorting (Ginger et al., 1998). Inactivation of the tipA gene also results in aberrant cell sorting (Stege et al., 1997). The tipA gene encodes a cytosolic protein, which is preferentially localized in pstO cells, suggesting that cell sorting can be influenced by intracellular signals.

How does DdCAD-1 affect cell sorting? It is likely that the effect of DdCAD-1 on cell sorting is an indirect one. The loss of DdCAD-1 expression may affect the expression and function of genes that can influence cell sorting. Potentially the precocious expression of gp80 at a higher level could account in part for the sorting phenotype in cadA cells. As the cadA cells accumulate more gp80 on the cell surface, they become more adhesive than wild-type cells (Fig. 9). After aggregation, cell movement continues within cell mounds, with the peripheral cells spiraling upwards to form the tip of the slug (Abe et al., 1994; Siegert and Weijer, 1995; Clow et al., 2000). Increased cell adhesiveness due to gp80 may repress cell motility and hinder the sorting out process. Indeed, prestalk cells fail to move forward to their anterior position in chimeric slugs (Fig. 7). Recently, we created a double knockout mutant (cadA/csaA). Neutral Red-stained cells of this mutant are capable of sorting to the anterior zone of the slug, suggesting that the gp80-null mutation is epistatic to the cadA mutation (E. Huang, W. F. L. and C.-H. Siu, unpublished).

Cell-cell adhesion has also been shown to regulate the size of aggregates and fruiting bodies (Kamboj et al., 1990; Roisin-Bouffay et al., 2000). The larger aggregate and slug sizes observed with the cadA cells are consistent with their elevated level of gp80 expression. The observation that the loss of DdCAD-1 enhances the expression of gp80 suggests that intercellular adhesion may be coupled to gene regulation. The synthesis of gp80 is highly augmented by cAMP pulses (Desbarats et al., 1992). As the formation of cell-cell contacts is known to affect cAMP metabolism and inhibit cAMP signaling (Fontana and Price, 1989; Fontana et al., 1991a; Fontana et al., 1991b), it is possible that the loss of DdCAD-1 expression may somehow enhance cAMP signaling and stimulate a higher level of gp80 expression. However, inhibition of cell-cell adhesion by either EDTA or carnitine leads to reduced levels of gp80 expression (Desbarats et al., 1994). These results suggest that the loss of DdCAD-1 expression and the inhibition of EDTA-sensitive cell adhesion sites may elicit different intracellular signals that can lead to opposite outcomes. It is also possible that the inhibition of gp80 expression by EDTA or carnitine is due to the inhibition of the Mg2+-dependent sites or the pleiotropic effects of these inhibitors.

Cell-type proportion is stringently regulated during Dictyostelium development, with ∼20% of the cells forming prestalk cells. This is a complex phenomenon, which probably requires the delicate balance among many factors (Mohanty and Firtel, 1999). In wild-type cells, the proportions of prestalk and prespore cells are kept relatively constant regardless the size of the slug. However, an increase in the number of prestalk cells is observed in slugs of cadA cells. Eventually, cadA cells produce taller fruiting bodies with smaller sori, with a 40% reduction in the yield of spores (Fig. 4). Although DdCAD-1 is still present in great abundance in the latter half of the developmental cycle, it is restricted mostly to the cytoplasm as a soluble protein (Sesaki and Siu, 1996). Our results thus implicate an intracellular function for DdCAD-1 in cell-type proportioning. The Ca2+-binding properties of DdCAD-1 raise the possibility that this protein is involved in the regulation of Ca2+ homeostasis during development. Cytosolic DdCAD-1 may be involved in the sequestration of Ca2+. Thus, the loss of DdCAD-1 in cadA cells can lead to higher levels of free Ca2+ in the cytosol. As high levels of free Ca2+ within cells have been associated with a propensity to prestalk cell differentiation (Newell et al., 1995; Saran et al., 1994; Cubitt et al., 1995; Azhar et al., 1996), the aberrant cell type proportioning seen in cadA cells may be related to increased levels of free Ca2+.

Fig. 1.

Construction of integration vector and isolation of cadA-null strains. (A) The 3.8 kb genomic DNA containing the cadA gene. The three black rectangles represent the three exons encoding DdCAD-1. Several unique restriction enzyme cut sites are also indicated. (B) The sequential steps involved in the construction of the cadA-null mutant strains. First, the genomic fragment containing the cadA gene was circularized and then cut at the HincII site. The linearized fragment was ligated to the SmaI site of the integration vector pbsrΔEco to generate pbsr/cadA. Next, the plasmid DNA was cut with EcoRI and used to transfect KAX3 cells by electroporation in the presence of excess EcoRI. Gene disruption was achieved by homologous recombination between the endogenous DNA and the plasmid DNA. (C) Protein blot analysis of blasticidin-resistant mutant clones. Total cell protein from 2×105 cells collected at 3 hours of development was separated by SDS-PAGE and the protein blots were probed with rabbit antibodies raised against DdCAD-1. (D) DNA blot analysis of the two cadA clones. Genomic DNA isolated from wild-type or cadA cells was digested with EcoRI and then separated by electrophoresis in a 1% agarose gel and the DNA blot was hybridized with either 32P-labeled cadA cDNA or pbsrΔEco DNA.

Fig. 1.

Construction of integration vector and isolation of cadA-null strains. (A) The 3.8 kb genomic DNA containing the cadA gene. The three black rectangles represent the three exons encoding DdCAD-1. Several unique restriction enzyme cut sites are also indicated. (B) The sequential steps involved in the construction of the cadA-null mutant strains. First, the genomic fragment containing the cadA gene was circularized and then cut at the HincII site. The linearized fragment was ligated to the SmaI site of the integration vector pbsrΔEco to generate pbsr/cadA. Next, the plasmid DNA was cut with EcoRI and used to transfect KAX3 cells by electroporation in the presence of excess EcoRI. Gene disruption was achieved by homologous recombination between the endogenous DNA and the plasmid DNA. (C) Protein blot analysis of blasticidin-resistant mutant clones. Total cell protein from 2×105 cells collected at 3 hours of development was separated by SDS-PAGE and the protein blots were probed with rabbit antibodies raised against DdCAD-1. (D) DNA blot analysis of the two cadA clones. Genomic DNA isolated from wild-type or cadA cells was digested with EcoRI and then separated by electrophoresis in a 1% agarose gel and the DNA blot was hybridized with either 32P-labeled cadA cDNA or pbsrΔEco DNA.

Fig. 2.

Loss of EDTA/EGTA-sensitive adhesion sites in cadA cells. Cells were developed in 17 mM phosphate buffer (pH 6.4) for 4 hours. Cell aggregates were dissociated mechanically and cell reassociation was assayed in the presence of 10 mM EDTA or 10 mM EGTA. (A) Reassociation of KAX3 in the presence of EDTA (▵) or EGTA (□), or in their absence (○). (B) Reassociation of cadA-10 cells in the presence of EDTA (▵) or EGTA (□), or in their absence (•). (C) Effects of soluble DdCAD-1 and anti-DdCAD-1 Fab on the reassociation of cadA-10 cells. Mutant cells were allowed to reassociate in the absence (•) or presence (▿)of 50 μg/ml soluble DdCAD-1 or 50 μg/ml anti-DdCAD-1 Fab (◊) or 20 mM carnitine (▴). Data represent mean±s.d. (n=3).

Fig. 2.

Loss of EDTA/EGTA-sensitive adhesion sites in cadA cells. Cells were developed in 17 mM phosphate buffer (pH 6.4) for 4 hours. Cell aggregates were dissociated mechanically and cell reassociation was assayed in the presence of 10 mM EDTA or 10 mM EGTA. (A) Reassociation of KAX3 in the presence of EDTA (▵) or EGTA (□), or in their absence (○). (B) Reassociation of cadA-10 cells in the presence of EDTA (▵) or EGTA (□), or in their absence (•). (C) Effects of soluble DdCAD-1 and anti-DdCAD-1 Fab on the reassociation of cadA-10 cells. Mutant cells were allowed to reassociate in the absence (•) or presence (▿)of 50 μg/ml soluble DdCAD-1 or 50 μg/ml anti-DdCAD-1 Fab (◊) or 20 mM carnitine (▴). Data represent mean±s.d. (n=3).

Fig. 3.

Developmental phenotype of cadA cells. Wild-type and cadA cells were developed on agar plates and aggregates were examined under a dissection microscope. KAX3 cells: (A) even splitting of aggregates; (B) finger structures derived from a single aggregate; (C) fruiting bodies. cadA-12 cells: (D) aggregates showing nodule-like structures protruding randomly from the surface (arrows), (E) elongated finger structures still carrying the nodule-like protrusions, often at the apical ends (arrow), and (F) fruiting bodies.

Fig. 3.

Developmental phenotype of cadA cells. Wild-type and cadA cells were developed on agar plates and aggregates were examined under a dissection microscope. KAX3 cells: (A) even splitting of aggregates; (B) finger structures derived from a single aggregate; (C) fruiting bodies. cadA-12 cells: (D) aggregates showing nodule-like structures protruding randomly from the surface (arrows), (E) elongated finger structures still carrying the nodule-like protrusions, often at the apical ends (arrow), and (F) fruiting bodies.

Fig. 4.

Effects of cadA gene inactivation on cell differentiation. Equal numbers of KAX3 cells and cadA cells were deposited on 2% non-nutrient agar for development. (A) Reduction in spore yields in cadA strains. Fruiting bodies were collected from the agar dish after 2 days of development and the total number of spores was estimated. The relative yield of spores was calculated. Data represent mean±s.d. of three experiments. (B) Increase in Neutral Red-strained cells in cadA slugs. KAX3 (wild-type) (▪) and cadA-10 cells (⧫) were labeled with Neutral Red and then deposited on 2% non-nutrient agar plates at 5×105 cells/cm2 for development in the dark at 22°C. Cells at different stages of development were collected and dissociated in 17 mM phosphate buffer (pH 6.4), containing 20 mM EDTA, and the percentage of Neutral Red-stained cells was determined. Data represent mean±s.d. of three experiments.

Fig. 4.

Effects of cadA gene inactivation on cell differentiation. Equal numbers of KAX3 cells and cadA cells were deposited on 2% non-nutrient agar for development. (A) Reduction in spore yields in cadA strains. Fruiting bodies were collected from the agar dish after 2 days of development and the total number of spores was estimated. The relative yield of spores was calculated. Data represent mean±s.d. of three experiments. (B) Increase in Neutral Red-strained cells in cadA slugs. KAX3 (wild-type) (▪) and cadA-10 cells (⧫) were labeled with Neutral Red and then deposited on 2% non-nutrient agar plates at 5×105 cells/cm2 for development in the dark at 22°C. Cells at different stages of development were collected and dissociated in 17 mM phosphate buffer (pH 6.4), containing 20 mM EDTA, and the percentage of Neutral Red-stained cells was determined. Data represent mean±s.d. of three experiments.

Fig. 5.

Aberrant cell sorting patterns in cadA slugs. (A-C) The distribution of prestalk and prespore cell in wild-type and mutant slugs was examined using cells stained with Neutral Red: (A) wild-type slugs; (B,C) cadA slugs. (D-H) The prestalk-prespore pattern was examined using GFP-expressing transformants. Strain JS24 (cotB::GFP; cadA+) was derived from KAX3 (cadA+) and strain TL144 (cotB::GFP; cadA) was derived from TL98 (cadA). (D) JS24 cells were developed on 2% agar for 20 hours and slugs showed the normal sorting pattern. (E) TL144 cells showed low levels of GFP expression at the mound stage and GFP-labeled cells were present in a nodule-like protrusion (arrowheads): phase micrograph (left panel); epifluorescence micrograph (right panel). (F) TL144 slug showing the non-sorted pattern. (G) TL144 slugs with an abnormally large anterior zone of unlabeled cells. (H) TL144 slugs showing aberrant sorting patterns. Scale bars: 1 mm.

Fig. 5.

Aberrant cell sorting patterns in cadA slugs. (A-C) The distribution of prestalk and prespore cell in wild-type and mutant slugs was examined using cells stained with Neutral Red: (A) wild-type slugs; (B,C) cadA slugs. (D-H) The prestalk-prespore pattern was examined using GFP-expressing transformants. Strain JS24 (cotB::GFP; cadA+) was derived from KAX3 (cadA+) and strain TL144 (cotB::GFP; cadA) was derived from TL98 (cadA). (D) JS24 cells were developed on 2% agar for 20 hours and slugs showed the normal sorting pattern. (E) TL144 cells showed low levels of GFP expression at the mound stage and GFP-labeled cells were present in a nodule-like protrusion (arrowheads): phase micrograph (left panel); epifluorescence micrograph (right panel). (F) TL144 slug showing the non-sorted pattern. (G) TL144 slugs with an abnormally large anterior zone of unlabeled cells. (H) TL144 slugs showing aberrant sorting patterns. Scale bars: 1 mm.

Fig. 6.

Quantitative analysis of sorting patterns exhibited by wild-type and cadA slugs. JS24 cells (cotB::GFP; cadA+) and TL144 cells (cotB::GFP; cadA) were developed on non-nutrient agar and slugs were observed by epifluorescence microscopy at 18 hours of development. Images were separated into three different categories for JS24 cells (solid bars) and TL144 cells (stippled bars). Slugs with a clearly demarcated anterior zone were scored as sorted, while those without a clearly demarcated anterior zone were scored as non-sorted. All other aberrant patterns were scored as partially sorted.

Fig. 6.

Quantitative analysis of sorting patterns exhibited by wild-type and cadA slugs. JS24 cells (cotB::GFP; cadA+) and TL144 cells (cotB::GFP; cadA) were developed on non-nutrient agar and slugs were observed by epifluorescence microscopy at 18 hours of development. Images were separated into three different categories for JS24 cells (solid bars) and TL144 cells (stippled bars). Slugs with a clearly demarcated anterior zone were scored as sorted, while those without a clearly demarcated anterior zone were scored as non-sorted. All other aberrant patterns were scored as partially sorted.

Fig. 7.

Cell sorting in chimeric slugs. Neutral Red-stained cadA prestalk cells were dissected from the anterior quarter of the cadA-12 slugs and wild-type prespore cells were isolated from the posterior half of KAX3 slugs. The two cell populations were randomly mixed at a 1:4 ratio (A, KAX3 prestalk cells: KAX3 prespore cells; B, cadA-12 prestalk cells: KAX3 prespore cells) for aggregation and slug formation on an agar surface. The length of the Neutral Red stained anterior zone was measured for each slug and the R values were calculated and then plotted against the frequency of occurrence. The data represent the average values of two experiments.

Fig. 7.

Cell sorting in chimeric slugs. Neutral Red-stained cadA prestalk cells were dissected from the anterior quarter of the cadA-12 slugs and wild-type prespore cells were isolated from the posterior half of KAX3 slugs. The two cell populations were randomly mixed at a 1:4 ratio (A, KAX3 prestalk cells: KAX3 prespore cells; B, cadA-12 prestalk cells: KAX3 prespore cells) for aggregation and slug formation on an agar surface. The length of the Neutral Red stained anterior zone was measured for each slug and the R values were calculated and then plotted against the frequency of occurrence. The data represent the average values of two experiments.

Fig. 8.

Cell sorting between cadA cells and wild-type cells. Cells were developed in liquid for 4 hours and then either cadA or wild-type cells were labeled with DiI. Equal number of wild-type and cadA cells were mixed and shaken for 6 hours. (A) A pair of phase and epifluorescence images showing a random mixture of labeled and unlabeled cadA cells. (B) A pair of phase and epifluorescence images showing cell sorting between labeled cadA cells and unlabeled KAX3 cells. Scale bars: 20 μm. (C) The percentage of aggregates showing cell sorting was scored for the different cell mixtures. The DiI-labeled cell strain is indicated by an asterisk.

Fig. 8.

Cell sorting between cadA cells and wild-type cells. Cells were developed in liquid for 4 hours and then either cadA or wild-type cells were labeled with DiI. Equal number of wild-type and cadA cells were mixed and shaken for 6 hours. (A) A pair of phase and epifluorescence images showing a random mixture of labeled and unlabeled cadA cells. (B) A pair of phase and epifluorescence images showing cell sorting between labeled cadA cells and unlabeled KAX3 cells. Scale bars: 20 μm. (C) The percentage of aggregates showing cell sorting was scored for the different cell mixtures. The DiI-labeled cell strain is indicated by an asterisk.

Fig. 9.

Expression of gp80 in cadA cells. KAX3 cells and cadA-12 cells were developed on agar and collected at different developmental stages. (A) Protein blots were probed with an anti-gp80 antibody. Equal amounts of proteins were loaded from each sample and the actin band of an identical gel stained with Coomassie Blue is included. (B) Cells at different developmental times were subjected to the cell cohesion assay in 5 mM EDTA. The percent cell aggregation was estimated after 45 minutes for KAX3 cells (•) and cadA-12 cells (○). The inhibitory effects of anti-gp80 mAb on cell reassociation were determined for cells at 8 hours and 10 hours of development. KAX3 cells (▪) and cadA-12 cells (□) at 2×107 cells/ml were preincubated with anti-gp80 mAb for 20 minutes at 4°C and then diluted 10-fold in 0.25 mg/ml of goat Fab against mouse IgG for the cell cohesion assay (Siu et al., 1985).

Fig. 9.

Expression of gp80 in cadA cells. KAX3 cells and cadA-12 cells were developed on agar and collected at different developmental stages. (A) Protein blots were probed with an anti-gp80 antibody. Equal amounts of proteins were loaded from each sample and the actin band of an identical gel stained with Coomassie Blue is included. (B) Cells at different developmental times were subjected to the cell cohesion assay in 5 mM EDTA. The percent cell aggregation was estimated after 45 minutes for KAX3 cells (•) and cadA-12 cells (○). The inhibitory effects of anti-gp80 mAb on cell reassociation were determined for cells at 8 hours and 10 hours of development. KAX3 cells (▪) and cadA-12 cells (□) at 2×107 cells/ml were preincubated with anti-gp80 mAb for 20 minutes at 4°C and then diluted 10-fold in 0.25 mg/ml of goat Fab against mouse IgG for the cell cohesion assay (Siu et al., 1985).

Fig. 10.

Restoration of wild-type phenotype by ectopic expression of DdCAD-1. cadA-12 cells were transfected with the cadA cDNA construct and transformants that expressed similar levels of DdCAD-1 (strains WT33 and WT94) were analyzed. (A) Protein blots showing the expression of DdCAD-1 (arrowhead) in transformants. (B) Cell cohesion was assayed at 4 hours for strains WT33 (□), WT94 (▪), cadA-12 (○) and KAX3 (broken line). (C) Protein blots showing changes in the level of gp80 expression during development. Equal amounts of proteins were loaded and the actin band of the Coomassie Blue stained gel is shown. (D) Cell cohesion assays were carried out at different stages of development for strains WT33 (□), KAX3 (•) and cadA-12 (○).

Fig. 10.

Restoration of wild-type phenotype by ectopic expression of DdCAD-1. cadA-12 cells were transfected with the cadA cDNA construct and transformants that expressed similar levels of DdCAD-1 (strains WT33 and WT94) were analyzed. (A) Protein blots showing the expression of DdCAD-1 (arrowhead) in transformants. (B) Cell cohesion was assayed at 4 hours for strains WT33 (□), WT94 (▪), cadA-12 (○) and KAX3 (broken line). (C) Protein blots showing changes in the level of gp80 expression during development. Equal amounts of proteins were loaded and the actin band of the Coomassie Blue stained gel is shown. (D) Cell cohesion assays were carried out at different stages of development for strains WT33 (□), KAX3 (•) and cadA-12 (○).

Fig. 11.

Cell type proportions in wild-type and transformed cell strains. Cells were pre-labeled with Neutral Red and development was carried out as described in the legend of Fig. 4. (A) The number of Neutral Red-stained cells was scored at 18 hours. (B) Spore yield was determined after 2 days. Data represent mean±s.d. (n=3).

Fig. 11.

Cell type proportions in wild-type and transformed cell strains. Cells were pre-labeled with Neutral Red and development was carried out as described in the legend of Fig. 4. (A) The number of Neutral Red-stained cells was scored at 18 hours. (B) Spore yield was determined after 2 days. Data represent mean±s.d. (n=3).

We thank Drs Reinhardt Reithmeier and David MacLennan for valuable advice, members of the Siu laboratory for discussion, and Tak Yee Lam and Eric Huang for expert technical support. This work was supported by Operating Grant MT-6140 from the Canadian Institutes of Health Research to C. H. S. and the National Science Foundation (MCB9728463) to W. F. L. E. Wong was supported by a Studentship from the Canadian Institutes of Health Research and J. Wang was supported in part by a Connaught Scholarship from the University of Toronto.

Abe, T.,Early, A., Siegert, F., Weijer, C. and Williams, J. (
1994
). Patterns of cell movement within the Dictyostelium slug revealed by cell type-specific, surface labeling of living cells.
Cell
77
,
687
-699.
Adachi, H., Hasebe, T., Yoshinaga, K., Ohta, T. and Sutoh, K. (
1994
). Isolation of Dictyostelium discoideum cytokinesis mutants by restriction enzyme-mediated integration of the blasticidin S resistance marker.
Biochem. Biophys. Res. Commun
.
205
,
1808
-1814.
Azhar, M., Manogaran, P. S., Kennady, P. K., Pande, G. and Nanjundiah, V. (
1996
). A Ca2+-dependent early functional heterogeneity in amoebae of Dictyostelium discoideum, revealed by flow cytometry.
Exp. Cell Res
.
227
,
344
-351.
Beug, H., Katz, F. and Gerisch, G. (
1973
). Dynamics of antigenic membranes sites relating to cell aggregation in Dictyostelium discoideum.
J. Cell Biol
.
56
,
647
-658.
Brar, S. K. and Siu, C.-H. (
1993
). Characterization of the cell adhesion molecule gp24 in Dictyostelium discoideum.
J. Biol. Chem
.
268
,
24902
-24909.
Clow, P. A., Chen, T., Chisholm, R. L. and McNally, J. G. (
2000
). Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion.
Development
127
,
2715
-2728.
Cubitt, A. B., Firtel, R. A., Fischer, G., Jaffe, L. F. and Miller, A. L. (
1995
). Patterns of free calcium in multicellular stages of Dictyostelium expressing jellyfish apoaequorin.
Development
121
,
2291
-2301.
Desbarats, L., Lam, T. Y., Wong, L. M. and Siu, C.-H. (
1992
) Identification of a unique cAMP-response element in the gene encoding the cell adhesion molecule gp80 in Dictyostelium discoideum.
J. Biol. Chem
.
267
,
19655
-19664.
Desbarats, L., Brar, S. K. and Siu, C.-H. (
1994
) Involvement of cell-cell adhesion in the expression of the cell cohesion molecule gp80 in Dictyostelium discoideum.
J. Cell Sci
.
107
,
1705
-1712.
Devine, K. and Loomis, W. F. (
1985
). Molecular characterization of anterior-like cells in Dictyostelium discoideum.
Dev. Biol
.
107
,
364
-372.
Dynes, J. L., Clark, A. M., Caulks, G., Cusp, A., Loomis, W. F. and Firtel, R. A. (
1994
). LagC is required for cell-cell interactions that are essential for cell-type differentiation in Dictyostelium.
Genes Dev
.
8
,
948
-958.
Early, A., Abe, T. and Williams, J. (
1995
). Evidence for positional differentiation of prestalk cells and for a morphogenetic gradient in Dictyostelium.
Cell
83
,
91
-99.
Edelman, G. M. and Crossin, K. L. (
1991
). Cell adhesion molecules: Implications for a molecular histology.
Annu. Rev. Biochem
.
60
,
155
-190.
Fontana, D. R. (
1993
). Two distinct adhesion systems are responsible for EDTA-sensitive adhesion in Dictyostelium discoideum.
Differentiation
53
,
139
-147.
Fontana, D. R. (
1995
). Dictyostelium discoideum cohesion and adhesion. In The Principles of Cell Adhesion (ed. P. D. Richardson and M. Steiners), pp. 63-86. Florida: CRC Press.
Fontana, D. R. and Price, P. L. (
1988
). Contact alters cAMP metabolism in aggregation-competent Dictyostelium amoebae.
Dev. Genet
.
9
,
279
-292.
Fontana, D. R., Luo, C. and Phillips, J. C. (
1991
a). Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.
Mol. Cell. Biol
.
11
,
468
-475.
Fontana, D. R., Price, P. L. and Phillips, J. C. (
1991
b). Cell-cell contact mediates cAMP secretion in Dictyostelium discoideum.
Dev. Genet
.
12
,
54
-62.
Fosnaugh, K. L. and Loomis, W. F. (
1993
) Enhancer regions responsible for temporal and cell-type-specific expression of a spore coat gene in Dictyostelium.
Dev. Biol
.
157
,
38
-48.
Gao, E. N., Shier, P. and Siu, C.-H. (
1992
). Purification and partial characterization of a cell adhesion molecule (gp150) involved in postaggregation stage cell-cell binding in Dictyostelium discoideum.
J. Biol. Chem
.
267
,
9409
-9415.
Gerisch, G. (
1961
). Zellfunktionen und Zellfunktionswechsel in der Entwicklung von Dictyostelium discoideum V. Stadienspezifische Zellkontaktbildung und ihre quantitative Erfassung.
Exp. Cell Res
.
25
,
535
-554.
Gerisch, G. (
1980
). Univalent antibody fragments as tools for the analysis of cell interactions in Dictyostelium.
Curr. Top. Dev. Biol
.
14
,
234
-270.
Ginger, R. S., Drury, L., Baader, C., Zhukovskaya, N. V. and Williams, J. G. (
1998
). A novel Dictyostelium cell surface protein important for both cell adhesion and cell sorting.
Development
125
,
3343
-3352.
Gumbiner, B. M. (
1996
). Cell adhesion: the molecular basis of tissue architecture and morphogenesis.
Cell
84
,
345
-357.
Harris, T. J. C., Awrey, D. E., Cox, B. J., Ravandi, A., Tsang, A. and Siu, C.-H. (
2001
a). Involvement of a Triton-insoluble floating fraction in Dictyostelium cell-cell adhesion.
J. Biol. Chem
.
276
,
18640
-18648.
Harris, T. J. C., Ravandi, A. and Siu, C.-H. (
2001
b). Assembly of glycoprotein-80 adhesion complexes in Dictyostelium.
J. Biol. Chem
.
276
,
48764
-48774.
Hynes, R. O. (
1999
). Cell adhesion: old and new questions.
Trends Cell Biol
.
9
,
M33
-37.
Jiang, Y., Levine, H. and Glazier, J. (
1998
). Possible cooperation of differential adhesion and chemotaxis in mound formation of Dictyostelium.
Biophys. J
.
75
,
2615
-2625.
Kamboj, R. K., Wong, L. M., Lam, T. Y. and Siu, C.-H. (
1988
). Mapping of a cell-binding domain in the cell adhesion molecule gp80 of Dictyostelium discoideum.
J. Cell Biol
.
107
,
1835
-1843.
Kamboj, R. K., Gariepy, J. and Siu, C.-H. (
1989
). Identification of an octapeptide involved in homophilic interaction of the cell adhesion molecule gp80 of Dictyostelium discoideum.
Cell
59
,
615
-625.
Kamboj, R. K., Lam, T. Y. and Siu, C.-H. (
1990
). Regulation of slug size by the cell adhesion molecule gp80 in Dictyostelium discoideum.
Cell Regul
.
1
,
715
-729.
Kellerman, K. A. and McNally, J. G. (
1999
). Mound-cell movement and morphogenesis in Dictyostelium.
Dev. Biol
.
15
,
416
-429.
Knecht, D. A., Fuller, D. L. and Loomis, W. F. (
1987
). Surface glycoprotein, gp24, involved in early adhesion of Dictyostelium discoideum.
Dev. Biol
.
121
,
277
-283.
Kuspa, A. and Loomis, W. F. (
1992
). Tagging developmental genes in Dictyostelium by restriction enzyme- mediated integration of plasmid DNA,
Proc. Natl. Acad. Sci. USA
89
,
8803
-8807.
Lam, T. Y., Pickering, G., Geltosky, J. and Siu, C.-H. (
1981
). Differential cell cohesiveness expressed by prespore and prestalk cells of Dictyostelium discoideum.
Differentiation
20
,
22
-28.
Loomis, W. F. (
1975
). Dictyostelium discoideum: A Developmental System. New York, Academic Press.
Mohanty, S. and Firtel, R. A. (
1999
). Control of spatial patterning and cell-type proportioning in Dictyostelium.
Semin. Cell Dev. Biol
.
10
,
597
-607.
Murray, B. A., Niman, H. L. and Loomis, W. F. (
1983
). Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum.
Mol. Cell. Biol
.
3
,
863
-870.
Newell, P. C. (
1995
). Signal transduction and motility of Dictyostelium.
Biosci. Rep
.
15
,
445
-462.
Nicol, A., Rappel, W., Levine, H. and Loomis, W. F. (
1999
). Cell-sorting in aggregates of Dictyostelium discoideum.
J. Cell Sci
.
112
,
3923
-3929.
Roisin-Bouffay, C., Jang, W., Caprette, R. D. and Gomer, H. R. (
2000
). A precise group size in Dictyostelium is generated by a cell-counting factor modulating cell-cell adhesion.
Mol. Cell
6
,
953
-959.
Saran, S., Azhar, M., Manogaran, P. S., Pande, G. and Nanjundiah, V. (
1994
). The level of sequestered calcium in vegetative amoebae of Dictyostelium discoideum can predict post-aggregative cell fate.
Differentiation
57
,
163
-169.
Sesaki, H. and Siu, C.-H. (
1996
). Novel redistribution of the Ca2+-dependent cell adhesion molecule DdCAD-1 during development of Dictyostelium discoideum.
Dev. Biol
.
177
,
504
-511
Sesaki, H., Wong, E. F. and Siu, C.-H. (
1997
). The cell adhesion molecule DdCAD-1 in Dictyostelium is targeted to the cell surface by a nonclassical transport pathway involving contractile vacuoles.
J. Cell Biol
.
138
,
939
-951.
Siegert, F. and Weijer, C. J. (
1995
). Spiral and concentric waves organize multicellular Dictyostelium mounds.
Curr. Biol
.
5
,
937
-943.
Siu, C.-H., Des Roches, B. and Lam, T. Y. (
1983
). Involvement of a cell surface glycoprotein in the cell sorting process of Dictyostelium discoideum.
Proc. Natl. Acad. Sci. USA
80
,
6596
-6600.
Siu, C.-H., Lam, T. Y. and Choi, A. H. (
1985
). Inhibition of cell-cell binding at the aggregation stage of Dictyostelium discoideum development by monoclonal antibodies directed against an 80,000-dalton surface glycoprotein.
J. Biol. Chem
.
260
,
16030
-16036.
Siu, C.-H., Cho, A. and Choi, A. H. C. (
1987
). The contact site A glycoprotein directly mediates cell-cell adhesion by homophilic interaction in Dictyostelium discoideum.
J. Cell Biol
.
105
,
2523
-2533.
Siu, C.-H., Harris, T. J. C., Wong, E. F. S., Yang, C., Sesaki, H. and Wang, J. (
1997
). Cell adhesion molecules in Dictyostelium. In Dictyostelium – A Model System for Cell and Developmental Biology (ed. Y. Maeda, K. Inouye and I. Takeuchi), pp. 111-121. Tokyo: Universal Academy Press.
Stege, J. T., Shaulsky, G. and Loomis, W. F. (
1997
). Sorting of the initial cell types in Dictyostelium is dependent on the tipA gene.
Dev. Biol
.
185
,
34
-41.
Stein, T. and Gerisch, G. (
1996
). Oriented binding of a lipid-anchored cell adhesion protein onto a biosensor surface using hydrophobic immobilization and photoactive crosslinking.
Anal. Biochem
.
237
,
252
-259
Steinberg, M. S. (
1975
). Adhesion-guided multicellular assembly: a commentary upon the postulates, real and imagined, of the differential adhesion hypothesis, with special attention to computer simulations of cell sorting,
J. Theor. Biol
.
55
,
431
-443.
Sukumaran, S., Brown, J. M., Firtel, R. A. and McNally, J. G. (
1998
). lagC-null and gbf-null cells define key steps in the morphogenesis of Dictyostelium mounds.
Dev. Biol
.
200
,
16
-26.
Sussman, M. (
1987
). Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions.
Methods Cell Biol
.
28
,
9
-29.
Sutoh, K. (
1993
). A transformation vector for Dictyostelium discoideum with a new selectable marker bsr.
Plasmid
30
,
150
-154.
Wang, J., Hou, L., Awrey, D., Loomis, W. F., Firtel, R. A. and Siu, C.-H. (
2000
). The membrane glycoprotein gp150 is encoded by the lagC gene and mediates cell-cell adhesion by heterophilic binding during Dictyostelium development.
Dev. Biol
.
227
,
734
-745.
Weijer, C. J., David, C. N. and Sternfeld, J. (
1987
). Vital staining methods used in the analysis of cell sorting in Dictyostelium discoideum.
Methods Cell Biol
.
28
,
449
-459.
Wong, E. F. S., Brar, S. K., Sesaki, H., Yang, C. and Siu, C.-H. (
1996
). Molecular cloning and characterization of DdCAD-1, a Ca2+-dependent cell-cell adhesion molecule, in Dictyostelium discoideum.
J. Biol. Chem
.
271
,
16399
-16408.
Wu, X. F., Kamboj, R. K, Gariepy, J. and Siu, C.-H. (
1992
). The 80L5C4 epitope overlaps with the homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium.
Biochem. Cell Biol
.
70
,
246
-249.
Yang, C., Brar, S. K., Desbarats, L. and Siu, C.-H. (
1997
). Synthesis of the Ca2+-dependent cell adhesion molecule DdCAD-1 is regulated by multiple factors during Dictyostelium development.
Differentiation
61
,
275
-284.