Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
Nat Protoc. 2009;4(11):1623-31. doi: 10.1038/nprot.2009.175. Epub 2009 Oct 15.
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.
小分子 GTPases 作为分子开关，调节多种细胞功能，如增殖、细胞运动和囊泡运输。基于荧光共振能量转移 (FRET) 原理的遗传编码生物传感器可以可视化活细胞中小 GTPases 的时空活性，从而帮助我们直观生动地理解小分子 GTPases 的作用。这里我们描述了使用 FRET 生物传感器进行活细胞成像的方案。有几种类型的 FRET 生物传感器；本方案侧重于由供体和受体荧光蛋白、小分子 GTPase、其结合伴侣以及（如有必要）细胞内定位信号组成的小分子 GTPase 的分子内或单分子 FRET 生物传感器。这些 FRET 生物传感器揭示了活细胞中小 GTPases 的时空活性，这是传统生化方法无法获得的。FRET 生物传感器的制备和细胞培养需要 6 天。成像和处理需要 3-4 天完成。
