1 Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada
2 Center for Molecular Genetics, Department of Biology, University of California at San Diego, La Jolla, CA 92037, USA
*Author for correspondence (e-mail: chi.hung.siu{at}utoronto.ca)
Accepted 16 May 2002
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SUMMARY |
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Key words: Cell-cell adhesion, Gene disruption, Cell-type differentiation, Pattern formation, Dictyostelium
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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Construction of integration vector and cell transformation
The integration vector pbsrBgl, 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 pbsr
Bgl was eliminated by blunt-end ligation. The resulting plasmid was named pbsr
Eco. A 3.8 kb EcoRI fragment containing the cadA gene (GenBank Accession Number, AF340153) was isolated from a genomic
gt10 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 pbsr
Eco to generate pbsr/cadA (Fig. 1A,B).
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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 pbsrEco DNA as probes for 18-20 hours at 42°C in 50% formamide and 5xSSC (20xSSC, 3 M NaCl and 0.3 M sodium citrate). The filters were washed for 30 minutes each, first at room temperature with 2xSSC, and then under more stringent conditions at 65°C in 2xSSC plus 1% SDS, and finally in 0.1xSSC 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 2x107 cells/ml. After 4 hours, cells were resuspended at
2.5x106 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 5x105 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 Bonners salt solution containing 10 mM NaCl, 10 mM KCl and 3 mM CaCl2. Cells were resuspended at 5x107 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 Bonners 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.
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RESULTS |
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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 pbsr
Eco (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 pbsr
Eco 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.
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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.
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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.
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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.
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DISCUSSION |
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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+.
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ACKNOWLEDGMENTS |
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