Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors.
Small GTPases 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 (FRET) can visualize a spatio-temporal activity of small GTPases in living cells, thereby helping us to understand the role of small GTPases intuitively and vividly. Here we describe protocols of live cell imaging with the FRET biosensors. There are several types of FRET biosensors; this protocol focuses on intramolecular or unimolecular FRET biosensors of small GTPases that are made up of donor and acceptor fluorescence proteins, a small GTPase, its binding partner, and, if necessary, a subcellular localization signal. These FRET biosensors uncover the spatio-temporal activity of the small GTPases in living cells, which could not be obtained by conventional biochemical methods. Preparation of FRET biosensors and cell culture takes 6 d. Imaging and processing take 3-4 d to complete.
Aoki
Kazuhiro
K
Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
Matsuda
Michiyuki
M
D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004815:Epidermal Growth Factor / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; C107953:RAC1 protein, human; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D020559:Monomeric GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D018631:ras Proteins