engleski

Visualization of activated GTPase Rac1A in living Dictyostelium cells

In pursuit of visualizing the activated form of the small GTPase Rac1A in living Dictyostelium cells, a fusion between CRIB domain of a Pak1 kinase and the yellow fluorescent protein (YFP) was constructed. Pak1-CRIB, or GBD for GTPase-binding domain, is known to bind specifically to activated, GTP-bound form of Rac GTPases. This construct, Pak1-CRIB-YFP, was expressed in wild-type Dictyostelium cells and its localization was monitored by laser scanning confocal microscopy in the course of cell migration, endocytosis and cytokinesis. During migration, Pak1-CRIB-YFP localizes to leading edges and pseudopod tips of moving vegetative and aggregation-competent cells. During phagocytosis and macropinocytosis, Pak1-CRIB-YFP localizes to phagocytic cups and crown-like cortical extensions that mediate uptake of fluid in Dictyostelium. Surprisingly, however, no enrichment of the construct was detected during mitotic cell division, neither at the cell poles, nor at the cleavage furrow. These results are in a sharp contrast with the localization of another Rac1A-binding protein DGAP1, an IQGAP-related protein which is enriched at the trailing end of migrating cells and at the cleavage furrow of dividing cells. Taken together, these findings indicate that small GTPase Rac1A is engaged in regulation of divergent signaling pathways leading to formation of cortical domains with different localizations and functional roles.

Dictyostelium; Rac1A; CRIB

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