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Disruption of the gene encoding the cell adhesion molecule DdCAD-1 leads to aberrant cell sorting and cell-type proportioning during Dictyostelium development

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

View larger version (41K): [in a new window]   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 pbsrEco 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 2x105 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 pbsrEco DNA.

View larger version (16K): [in a new window]   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).

View larger version (91K): [in a new window]   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.

View larger version (14K): [in a new window]   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 5x105 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.

View larger version (80K): [in a new window]   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.

View larger version (29K): [in a new window]   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.

View larger version (11K): [in a new window]   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.

View larger version (50K): [in a new window]   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.

View larger version (25K): [in a new window]   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 2x107 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).

View larger version (40K): [in a new window]   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 ().

View larger version (16K): [in a new window]   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).

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