Rap1 Overexpression Reveals That Activated RasD Induces Separable Defects duringDictyosteliumDevelopment

Abstract

One of theDictyostelium rasgenes,rasD,is expressed preferentially in prestalk cells at the slug stage of development and overexpression of this gene containing a G12T activating mutation causes the formation of aberrant multitipped aggregates that are blocked from further development (Reymondet al.,1986,Nature,323, 340–343). The ability of theDictyostelium rap1gene to suppress this abnormal developmental phenotype was investigated. Therap1gene and G12V activated and G10V negative mutant forms of therap1gene were independently linked to therasDpromoter and each construct used to transform M1, aDictyosteliumcell line expressing RasD[G12T]. Transformants of M1 that expressed Rap1 or Rap1[G12V] protein still formed multitipped aggregates, but most tips were able to complete development and form fruiting bodies. Cell lines showing this modified phenotype were designated ME (multitipped escape). Therap1[G10V] construct did not modify the M1 phenotype. These data suggest that overexpression of RasD[G12T] has two effects, the formation of a multitipped aggregate and a block in subsequent differentiation and that the expression of Rap1 or Rap1[G12V] reverses only the latter. Differentiation of ME cells in low density monolayers showed the identical low level of stalk and spore cell formation seen for M1 cells under the same conditions. Thus the cell autonomous defect in monolayer differentiation induced in the M1 strain was not corrected in the ME strain. Cell type-specific gene expression during the development of M1 cells is dramatically altered: prestalk cell-specific gene expression is greatly enhanced, whereas prespore-specific gene expression is almost suppressed (Louiset al.,1997,Mol. Biol. Cell,8, 303–312). During the development of ME cells,ecmA mRNA levels were restored to those seen for Ax3, andtagB mRNA levels were also markedly reduced, although not to Ax3 levels.cotCexpression in ME cells was enhanced severalfold relative to M1, although levels were still lower than those observed during the development of Ax3. The low expression ofcar1mRNA during early development of the M1 strain remained low during the development of ME cells. These data are consistent with the idea that the expression of RasD[G12T] affects two independent and temporally separated events and that only the later defect is reversed byrap1.