engleski

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

Rac proteins are the only canonical monomeric Rho GTPases in Dictyostelium, where they act as key regulators of the actin cytoskeleton. In order to monitor the dynamics of activated Rac1 proteins in migrating Dictyostelium cells, a fluorescent probe was developed that specifically binds the GTP-bound form of Rac1. The probe consists of a yellow fluorescent protein (DYFP) fused C-terminally to a GTPase-binding domain (GBD) from rat PAK1 kinase. PAK1_GBD was chosen on the basis of its specific interactions with Rac1A and Rac1C in yeast two-hybrid and GST pull-down screens. An interaction between PAK1_GBD and activated Rac1A was corroborated in living cells by fluorescence resonance energy transfer. In migrating cells PAK1_GBD-DYFP becomes transiently strongly enriched at the leading edge during protrusion of a pseudopod and localizes to endocytotic cups during phagocytosis and macropinocytosis. Activated Rac1 proteins therefore appear to participate in signaling pathways that control de novo actin polymerization at the protruding regions of Dictyostelium cell. Another effector of Rac1 GTPases, the IQGAP-related protein DGAP1, sequesters active Rac1 into a quaternary complex with actin-binding proteins cortexillins 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-DYFP strictly depends on the integrity of the actin cytoskeleton, whereas cortical localization of mRFPmars-DGAP1 does not. Taken together, these results suggest that Rac1 GTPases play important role both at the anterior and the posterior regions of polarized and migrating Dictyostelium cells.

Rho GTPases; actin cytoskeleton; Dictyostelium

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