engleski

Monitoring the graded Rac1 activity in Dictyostelium cells

Small Rho GTPases are major regulators of the actin cytoskeleton dynamics in eukaryotic cells. Usual approaches to examine their activity in living cells, such as fluorescence resonance energy transfer (FRET), are of limited use in rapidly moving cells, because short time available for image acquisition often results in an unacceptably low signal-to-noise ratio. Here, we describe characterization of a new fluorescent probe that selectively binds to active forms of Dictyostelium discoideum Rac1 GTPases, and demonstrate its excellent properties for live cell imaging. The probe is based on the GTPase-binding domain (GBD) from DPAKa kinase, and was selected on the basis of yeast two-hybrid screen, GST pull-down assay, and FRET measurements in live cells by fluorescence lifetime imaging microscopy (FLIM). The probe binds specifically to active Rac1 and has a low cytoplasmic background, thus enabling quantitative determination of the Rac1 activity in the cell membrane over an order of magnitude. Finally, overexpression of DPAKa(GBD)-DYFP probe induces no adverse effects on cell motility, cytokinesis and growth, thus enabling long-term imaging with negligible photobleaching and phototoxicity.

Dictyostelium discoideum; Rac1; biosensor; actin

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