engleski

A dual role for Rac1 GTPases in the regulation of cell motility

Rac proteins are the only canonical Rho family GTPases in Dictyostelium, where they act as key regulators of the actin cytoskeleton. In order to monitor the dynamics of activated Rac1 in Dictyostelium cells, a fluorescent probe was developed that specifically binds to GTP-bound form of Rac1. The probe is based on the GTPase- binding domain (GBD) from PAK1 kinase, and was selected on the basis of yeast two-hybrid, GST pull-down and fluorescence resonance energy transfer assays. PAK1_GBD localizes to leading edges of migrating cells and to endocytotic cups. As in vertebrates, activated Rac1 therefore appears to control de novo actin polymerization at protruding regions of the cell. Additionally, we found that the IQGAP-related protein DGAP1, which sequesters active Rac1 into a quaternary complex with actin-binding proteins cortexillin I and II, localizes to the trailing regions of migrating cells. Notably, PAK1_GBD and DGAP1, which both bind to Rac1-GTP, display mutually exclusive localizations in cell migration, phagocytosis and cytokinesis, and opposite dynamics of recruitment to the cell cortex upon stimulation with chemoattractants. Moreover, cortical localization of PAK1_GBD depends on integrity of the actin cytoskeleton, whereas cortical localization of DGAP1 does not. Taken together, these results imply that Rac1 GTPases play a dual role in regulation of cell motility and polarity in Dictyostelium.

Rac1; cell motility; cell polarization; FRET; actin cytoskeleton; Rho GTPases; dictyostelium

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