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Rho family G proteins including Rac regulate a variety of cellular functions, such as morphology, motility, and gene expression. Here we developed a fluorescence resonance energy transfer-based analysis in which we could monitor the activity of Rac1. To detect fluorescence resonance energy transfer, yellow fluorescent protein fused Rac1 and cyan fluorescent protein fused Cdc42-Rac1-interaction-binding domain of Pak1 protein were used as intermolecular probes of FRET. The fluorophores were separated with linear unmixing method. The fluorescence resonance energy transfer efficiency was measured by acceptor photobleaching assisted assay. With these methods, the Rac1 activity was visualized in a cell. The present findings indicate that this approach is sensitive enough to achieve results similar to those from ratiometric fluorescence resonance energy transfer analysis.

Visualization of the Activity of Rac1 Small GTPase in a Cell