Regulatory role of the Gα1 subunit in controlling cellular morphogenesis in Dictyostelium

Abstract

To determine the function of the Dictyostelium Gα1 subunit during aggregation and multicellular development, we analyzed the phenotypes of gα1 null cells and strains overexpressing either wild-type Gα1 or two putative constitutively active mutations of Gα1. Strains overexpressing the wild-type or mutant Gα1 proteins showed very abnormal culmination with an aberrant stalk differentiation. The similarity of the phenotypes between Gα1 overexpression and expression of a putative constitutively active Gα1 subunit suggests that these phenotypes are due to increased Gα1 activity rather than resulting from a non-specific interference of other pathways. In contrast, gα1 null strains showed normal morphogenesis except that the stalks were thinner and longer than those of wild-type culminants. Analysis of cell-type-specific gene expression using lacZ reporter constructs indicated that strains overexpressing Gα1 show a loss of ecmB expression in the central core of anterior prestalk AB cells. However, expression of ecmB in anterior-like cells and the expression of prestalk A-specific gene ecmA and the prespore-specific gene SP60/cotC appeared normal. Using a Gα1/lacZ reporter construct, we show that Gα1 expression is cell-type-specific during the multicellular stages, with a pattern of expression similar to ecmB, being preferentially expressed in the anterior prestalk AB cells and anterior-like cells. The developmental and molecular phenotypes of Gα1 overexpression and the cell-type-specific expression of Gα1 suggest that Gα1-mediated signaling pathways play an essential role in regulating multicellular development by controlling prestalk morphogenesis, possibly by acting as a negative regulator of prestalk AB cell differentiation. During the aggregation phase of development, gα1 null cells display a delayed peak in cAMP-stimulated accumulation of cGMP compared to wild-type cells, while Gα1 overexpressors and dominant activating mutants show parallel kinetics of activation but decreased levels of cGMP accumulation compared to that seen in wild-type cells. These data suggest that Gα1 plays a role in the regulation of the activation and/or adaptation of the guanylyl cyclase pathway. In contrast, the activation of adenylyl cyclase, another pathway activated by cAMP stimulation, was unaf fected in gα1 null cells and cell lines overexpressing wild-type Gα1 or the Gα1(Q206L) putative dominant activating mutation. However, the Gα1(G45V) putative constitutively active mutation showed significantly reduced adenylyl cyclase activity in response to cAMP. All Gα1 mutant cell lines aggregated normally; however, aggregates of cells expressing Gα1(G45V) developed ring-like structures that then developed a polarity and a small mound-like structure before forming a slug. Immunoprecipitation results suggest that the G45V phenotypes may be due to altered properties of this subunit and its association with the βg subunit.