engleski

A dual role for Rac1 GTPase 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 and GST pull-down screens. An interaction between PAK1_GBD and activated Rac1 was corroborated in living cells by fluorescence resonance energy transfer (FRET). In moving Dictyostelium cells, PAK1_GBD is strongly enriched at the leading edge where it co-localizes with F-actin, and it also localizes to endocytotic cups during phagocytosis and macropinocytosis. As in vertebrates, activated Rac1 therefore appears to participate in signalling pathways that control de novo actin polymerization at protruding regions of the cell. Additionally, the IQGAP-related protein DGAP1 sequesters active Rac1 into a quaternary complex with the actin-binding proteins cortexillin I and II and, notably, this complex localizes to the trailing, retracting regions of migrating cells. As assessed by latrunculin B treatment, cortical localization of PAK1_GBD strictly depends on the integrity of the actin cytoskeleton, whereas cortical localization of DGAP1 does not. Taken together, these results imply that Rac1 GTPases play a dual role, both at the front and in the back, in migrating Dictyostelium cells.

Rac1 ; cell motility ; actin cytoskeleton

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