engleski

Localization of a fluorescent probe for activated Rac1A in D. discoideum

Rac proteins are members of a broad family of monomeric Rho GTPases that act as key regulators of the actin cytoskeleton. We are using genetically tractable and highly motile Dictyostelium discoideum cells to study localization, dynamics and activity of Rac1A in processes that involve actin remodeling. To gain insight into its role in D. discoideum, we created a fluorescent probe that specifically binds the active form of Rac1A. Since Dictyostelium Rac GTPases are strongly related to each other, we 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, it localized to the endocytotic cup. During cytokinesis the probe didn’ t show any prominent localization.

Rho GTPases; actin cytoskeleton; Dictyostelium

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