Fuctional Analysis Of Cell Adhesion Molecules LagC/gp150and CadA/DdCAD-1During The Development Of Dictyostelium Discoideum

Cell adhesion molecule gp150plays an important role in post-aggregation stage of Dictyostelium discoideum development. The protein localization using antibodies against gpl50showed that the distribution pattern of gpl50varies during the various stages of development. In stream stage before aggregation, gpl50distributed uniformly in cytoplasm, after that, gp150occupied the periphery region of multicellular aggregates in mound stage. During the slug stage of development, the expression level of gp150in prestalk cells was much higher than that in prespore cells. In fruiting body stage, gpl50was enriched on the sporoderm of mature spores. High performance liquid chromatography was used to analyze PKA activity in wide-type KAx-3cells and gp150over expressed strain KAx-3(act15::lagC), and the results showed that, at the early stage of development (before10h development), PKA had lower activity in KAx-3cells than that in KAx-3(act15::lagQ cells. However, at the later stage of development, since the rapid accumulation of gp150, PKA showed higher activity in wide type cells than that inî™'Ax-3(act15::lagC) cells. These results suggested that there might be a feed back loop interaction between gp150and PKA, by which the two signal proteins might cooperate and regular the growth and development of D. discoideum.Cell adhesion molecule cadA/DdCAD-1is the first expressed adhesion molecule during the development of D. discoideum. To investigate the role of DdCAD-1in D. discoideum development, cadA mutant strain cadA-cells were colored by Natural Red, slug structure show obviously aberrant cell differentiation of prespore and prestalk cells. Exogenous recombinant protein His6-DdCAD-1can rescued this phenomenon. Moreover, recombinant protein His6-DdCAD-1can also rescue the decreasing spore yield of cadA-cells. When DdCAD-1expressed cells were mixed with cadA-cells, DdCAD-1expressed cells occupied the tips and posterior region of the chimeric culmination structures, which do not belong to spore region and would die eventually. All these results indicate that DdCAD-1plays a crucial role in cell differentiation and cell fate determination.In D. discoideum, soluble cell adhesion molecule DdCAD-1regulates cell-cell interaction through the interaction with an unknown anchoring protein on the plasma membrane. Far western blot analysis using different probes conjugated to DdCAD-1reveals that the potential DdCAD-1interacting protein was appeared between64kDa-98kDa. To isolate and identify the anchoring protein, GST-DdCAD-1and anchoring protein were cross linked by chemical cross-linker DSP and stable complex formed was isolated by co-immunoprecipitation with antibodies and protein A beads. Mass spectrometric analysis of the complex showed that the putative anchoring protein may belong to ATP-binding cassette transporter family.DdCAD-1is a Ca2+-dependent cell adhesion molecule expressed at the onset of Dictyostelium development. To investigate the multifaceted role of DdCAD-1, chimeras made up of cadA-cells and the wild-type parental AX4cells were analyzed. Results showed that cadA-cells were present predominantly in the central region surrounded by AX4cells, suggesting that the mutant cells might have migrated faster than AX4cells. In migration assays, AX4cells, cadA-cells and rescued cells exhibited similar migration rates at the vegetative stage. However, at4h of development, the cadA-cells migrated at a rate>2-fold higher than that of wildtype and rescued cells. Significantly, the addition of exogenous recombinant DdCAD-1to cadA-cells retarded their migration rate. Also, the cadA-cells attached less strongly to substratum than wildtype and rescued cells. Confocal microscopy revealed DdCAD-1on the ventral surface of cells, suggesting interactions between DdCAD-1and the substratum. Time-lapse microscopy of cell streams showed that the knockout cells migrated～1.7-fold faster than AX4cells. Both types of cells continued to move and rotate after their entry into the mound and the cadA-cells eventually sorted out from the AX4cells to form a core structure which gradually moved to the posterior region of the slug. The data suggest that the relative strengths of DdCAD-1-mediated cell-cell and cell-substratum interactions exert an influence on cell migration, which in turn modulates the special cell pattern formation and leads to anti-green beard effect.