Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors

Small GTPTPases act as molecular switches that regulate a variety of cellular functions, such as proliferation, cell movement and vesicle trafficking. Genetically encoded biosensors based on the principle of fluorescence resonance energy transfer (FRETRETRET) can visualize a spatio-temporal activity of small GTPTPases in living cells, thereby helping us to understand the role of small GTPTPases intuitively and vividly. Here we describe protocols of live cell imaging with the FRETRETRET biosensors. There are several types of FRETRETRET biosensors; this protocol focuses on intramolecular or unimolecular FRETRETRET biosensors of small GTPTPases that are made up of donor and acceptor fluorescence proteins, a small GTPTPase, its binding partner, and, if necessary, a subcellular localization signal. These FRETRETRET biosensors uncover the spatio-temporal activity of the small GTPTPases in living cells, which could not be obtained by conventional biochemical methods. Preparation of FRETRETRET biosensors and cell culture takes 6 d. Imaging and processing take 3-4 d to complete.