A RAS GAP IS ESSENTIAL FOR CYTOKINESIS AND SPATIAL PATTERNING IN DICTYOSTELIUM

Using the yeast two-hybrid system, we have identified developmentally regulated Dictyostelium genes whose encoded proteins interact with Ras . GTP but not Ras . GDP. By sequence homology and biochemical functio n, one of these genes encodes a Ras GAP (DdRasGAP1). Cells carrying a DdRasGAP1 gene disruption (ddrasgap1 null cells) have multiple, very d istinct growth and developmental defects as elucidated by examining th e phenotypes of ddrasgap1 null strains. First, vegetative ddrasgap1 nu ll cells are very large and highly multinucleate cells when grown in s uspension, indicating a severe defect in cytokinesis. When suspension- grown cells are plated in growth medium on plastic where they attach a nd can move, the cells rapidly become mono- and dinucleate by traction -mediated cell fission and continue to grow vegetatively with a number of nuclei (1-2) per cell, similar to wild-type cells. The multinuclea te phenotype, combined with results indicating that constitutive expre ssion of activated Ras does not yield highly multinucleate cells and d ata on Ras null mutants, suggest that Ras may need to cycle between GT P- and GDP-bound states for proper cytokinesis. After starvation, the large null tells undergo rapid fission when they start to move at the onset of aggregation, producing mononucleate cells that form a normal aggregate. Second, ddrasgap1 null cells also have multiple development al phenotypes that indicate an essential role of DdRasGAP1 in controll ing cell patterning. Multicellular development is normal through the m id-slug stage, after which morphological differentiation is very abnor mal and no culminant is formed: no stalk cells and very few spores are detected. lacZ reporter studies show that by the mid-finger stage, mu ch of the normal cell-type patterning is lost, indicating that proper DdRasGAP1 function and possibly normal Ras activity are necessary to m aintain spatial organization and for induction of prestalk to stalk an d prespore to spore cell differentiation. The inability of ddrasgap1 n ull cells to initiate terminal differentiation and form stalk cells is consistent with a model in which Ras functions as a mediator of inhib itory signals in cell-type differentiation at this stage. Third, DdRas GAP1 and cAMP dependent protein kinase (PKA) interact to control spati al organization within the organism. Overexpression of the PKA catalyt ic subunit in ddrasgap1 cells yields terminal structures that are mult iply branched but lack spores. This suggests that RasGAP and PKA may m ediate common pathways that regulate apical tip differentiation and or ganizer function, which in turn control spatial organization during mu lticellular development. It also suggests that DdRasGAP1 either lies d ownstream from PKA in the prespore to spore pathway or in a parallel p athway that is also essential for spore differentiation. Our results i ndicate that DdRasGAP1 plays an essential role in controlling multiple , potentially novel pathways regulating growth and differentiation in Dictyostelium and suggest a role for Ras in these processes.