engleski

How to track active Rac1 in live Dictyostelium cells?

Small Rho GTPases are major regulators of the actin cytoskeleton dynamics in eukaryotic cells. Common approaches for examination of their activity in living cells include probes based on fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC) and photoactivation. However, these methods might be of limited use, because short time available for image acquisition often leads to a low signal-to-noise ratio. Attempts to overcome this effect by increasing the intensity of illumination are restricted by photobleaching of probes and the cell’s photosensitivity. Here, we describe characterization of a new fluorescent probe that selectively binds to active forms of Dictyostelium Rac1 GTPases, and demonstrate its superior 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 by fluorescence lifetime imaging microscopy (FLIM). It binds specifically to active Rac1 which is located at the cell membrane and has a low cytoplasmic background. Overexpression of DPAKa(GBD)-DYFP probe induces no adverse effects on cell motility as estimated by a quantitative cell migration assay. In the end, its finely tuned intensity distribution enables quantitative measurements of the Rac1 activity in different parts of the cell membrane.

Dictyostelium discoideum; Rac1; biosensor

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