engleski

Monitoring the Rac1-mediated regulation of cell polarity in Dictyostelium discoideum

Dictyostelium cells are capable of completely reversing their polarity within 20 seconds, and thus undergo the fastest spontaneous re-polarization among eukaryotic cells. Signalling by small Rho GTPases plays an essential role in this process. Biosensors are therefore needed that are able to match these fast dynamics and enable protracted imaging at high recording rates. Rac proteins are the only Rho GTPases present in Dictyostelium. We have shown previously that Rac1 GTPases play a dual role in the regulation of actin assembly, which is a key determinant of cell polarity. At the cell front, Rac1A activates the Scar/WAVE complex and thereby stimulates the Arp2/3-mediated actin polymerization. At the cell back, Rac1A regulates stability of the cell cortex by initiating formation of a complex containing IQGAP-related proteins and the cortexillin heterodimer. Here, we describe selection and characterization of a new fluorescent probe that selectively binds to active forms of Dictyostelium Rac1 GTPases, and demonstrate its excellent properties for live cell imaging. The probe consists of a GBD domain from DPAKa kinase fused with a yellow fluorescent protein, and was selected on the basis of yeast two-hybrid screen, GST pull-down assay, and FRET measurements by fluorescence lifetime imaging microscopy (FLIM). The DPAKa(GBD)-DYFP probe binds specifically to active Rac1 GTPases, which are enriched at the anterior cell membrane during cell migration, and has a low cytoplasmic background. Importantly, expression of DPAKa(GBD)-DYFP induces no adverse effects on cell motility, cytokinesis and growth, thus enabling long-term imaging with negligible photobleaching and phototoxicity.

Dictyostelium discoideum; Rac1; biosensor; cell polarity

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