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. To monitor the dynamics of activated Rac1 in Dictyostelium cells, a fluorescent probe was developed that specifically binds to the 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. The PAK1 GBD localizes to leading edges of migrating cells and to endocytotic cups. Similarly to its role in vertebrates, activated Rac1 therefore appears to control de novo actin polymerization at protruding regions of the Dictyostelium 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 cortexillin 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 the PAK1 GBD 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 in regulation of cell motility and polarity in Dictyostelium.
Ruder Bošković Institute, Division of Molecular Biology, Bijenička 54, HR-10000 Zagreb,  Croatia.
Vedrana
V
Filić
Maja
M
Marinović
Jan
J
Faix
Igor
I
Weber
D008841:Actin Cytoskeleton / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line; D002465:Cell Movement; D000242:Cyclic AMP / Q000378:metabolism; D004023:Dictyostelium / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D005492:Folic Acid / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D053778:Thiazolidines / Q000494:pharmacology; D054462:p21-Activated Kinases / Q000737:chemistry / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000032:analysis / Q000378:metabolism / Q000502:physiology*
D019086:Bridged Bicyclo Compounds, Heterocyclic; D005456:Fluorescent Dyes; D020690:GTPase-Activating Proteins; D053778:Thiazolidines; D005492:Folic Acid; D000242:Cyclic AMP; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein; C037068:latrunculin B