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The effects of small GTP-binding proteins, Ras and Rap, on Dictyostelium Discoideum growth and differentiation

University of British Columbia

It had been previously demonstrated that the expression of an activated rasD gene in Dictyostelium Ax3 cells (designated [Gl2T]rasD strain) resulted in formation of aggregates with multi-tips instead of the normal single tips, and a block in further development (Reymond et al., 1986, Nature 323:340-343). To investigate the role of activated RasD[G12T] during Dictyostelium development, cell type specific gene expression was examined in the [G12T]nzsD strain and found to be dramatically deregulated. The expression of prestalk cell specific genes ecmA and tagB was markedly enhanced, while the expression of the prespore cell specific gene cotC was reduced to low levels. By using a reporter gene linked to a prestalk cell specific promoter (ecmA/lacZ) to monitor the fate of cells in the multi-tipped aggregate, it appeared that most of the cells eventually adopted prestalk gene expression characteristics. When [G12T]rasD and wild type Ax3 cells were mixed and then induced to differentiate, chimeric pseudoplasmodia were not formed. Thus the defect in [G12T]rasD cells could not be corrected by mixing with wild type cells. Both stalk and spore cell formation could be induced in low cell density monolayers of the [G12T]rasD strain, suggesting that at least part of the inhibition of stalk and spore formation during development of the multi-tipped aggregates involved inhibitory cell interactions within the cell mass. Since it has been shown that Rap proteins are capable of antagonizing the effects of activated Ras proteins in mammalian cell lines, the ability of Dictyostelium rapl' to block the abnormal developmental phenotype of the [G12T]rasD strain was investigated. The rapl gene, a G12V activated and G10V negative mutant forms of rapl were independently linked to the rasD promoter and each construct used to transform [G12T]rasD strain. Transformants that expressed Rapl or Rapl[G12V] protein and maintained high levels of RasD[G12T], exhibited a modified phenotype. They still formed multi-tipped aggregates but most tips were able to complete development and form fruiting bodies. This phenotype was designated Multi-tipped Escape (ME). The rapl [G10V] construct did not modify the multi-tipped phenotype. Analysis of cell type specific gene expression in the ME cells showed that ecmA and tagB mRNA levels, which were enhanced in the [G12T]rasD strain, were restored to the levels seen in wild type cells. Furthermore, cotC mRNA which was dramatically lowered in the [G12T]rasD strain, was partially restored in the ME strain. However, the low expression of carl mRNA levels observed during the early development of [G12T]rasD strain was not restored by the overexpression of Rapl. There was also no increase in stalk and spore cell formation in monolayers of ME transformant compared to the [G12T]rasD strain. These data suggest that RasD[G12T] had two temporally separated effects during development, an early and a late defect, rapl appeared to only rescue the late defect in the [G12T]rasD strain, and the phenotype and gene expression data of the ME transformants were consistent with this idea. rapl appeared to have a general ability to antagonize the effects of activated ras genes in Dictyostelium since it also affected the phenotype of a strain overexpressing a growth phase dependent activated ras, rasG[G12T]. The overexpression of the rapl gene in the RasG-G12T cells partially suppressed the defect in aggregation and the cytoskeletal defect in these cells, but did not affect the cytokinesis defect, suggesting that the cytokinesis defect was distinct from the other two defects. The partial rescue in carl mRNA expression by Rapl, suggested that carl repression did not fully account for the aggregation deficiency in RasG-G12T cells.

Thesis/Dissertation

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2009-03-16

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http://hdl.handle.net/2429/6059

Microbiology and Immunology

University of British Columbia

UBCV

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