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A novel fluorescent probe for activated RacA1 proteins in Dictyostelium discoideum

Rho family GTPases are monomeric G-proteins that act as key regulators of the actin cytoskeleton. In the nervous system, Rho family GTPases play an important role in neuronal growth cone motility, axonal migration, and dendritic spine morphogenesis. We are using genetically tractable and highly motile cells of the protist Dictyostelium discoideum to study localization, dynamics and activity of Rac proteins in processes that involve remodeling of the actin cytoskeleton. To gain insight into the role of Rac1A in D. discoideum, we created a fluorescent probe that specifically binds an active form of this small GTPase. Because Dictyostelium Rac GTPases are strongly related to each other, we first employed yeast two hybrid (Y2H) assay to test the interaction between 4 potential binding partners and 11 Rac proteins. In this way, we could pinpoint the interactor specific for active Rac1A. We tested an IQGAP-related protein DGAP1, which binds active Rac1A within a tetrameric cortical complex, and its GTPase-binding domain (GBD). We also tested regulatory domains of two other Rac downstream effectors that contain CRIB (Cdc/Rac interactive binding) motif: PBD (p21-binding domain) from Dictyostelium PAKa kinase and GBD from rat PAK1 kinase. Only GBD from rat Pak1 interacted under stringent conditions in Y2H assay with Rac1A. Moreover, it was the only partner that did not interact with the constitutively inactive form of Rac1A. This interaction was confirmed by GST pull-down assay. Therefore, PAK1_GBD was fused N-terminally to YFP and expressed in wild type cells. In non-motile cells, our probe was strongly enriched throughout the cortex, while in motile cells it always localized to the leading edge. In phagocytosis and macropinocytosis the probe localized to the endocytotic cup, whereas it didn’ t show any prominent localization during cytokinesis. We will also report on the development of a probe designed to monitor activity of Rac1A in situ, which is based on fluorescence resonance energy transfer (FRET).

Rac1A; GTPases; Dictyostelium; CRIB; FRET

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