Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors
Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors
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DOI：
10.1073/pnas.0807537105
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发表时间：
2008-09-23
影响因子：
11.1
通讯作者：
Wang, Yingxiao
中科院分区：
文献类型：
--
作者：
Ouyang, Mingxing;Sun, Jie;Wang, Yingxiao
基于荧光共振能量转移（FRET）的基因编码生物传感器使得在活细胞中以高时空分辨率对信号事件进行可视化成为可能。然而，这些生物传感器有限的灵敏度阻碍了它们在生物学研究中的广泛应用。我们将增强型青色荧光蛋白（ECFP）与黄色荧光蛋白的一种变体YPet进行配对。这种ECFP/YPet荧光共振能量转移对显著提高了生物传感器对多种信号分子的灵敏度（灵敏度提高数倍，且无需对每个单独的生物传感器进行定制优化），这些信号分子包括酪氨酸激酶Src、小GTP酶Rac、钙离子以及一种膜结合型基质金属蛋白酶MT1 - MMP。这些改进后的生物传感器的应用表明，在微图案化表面上的定向细胞迁移过程中，血小板源性生长因子（PDGF）对Src和Rac的激活呈现出不同的亚细胞模式。Rac的活性高度极化并集中在前沿，而Src的活性则相对均匀。这些荧光共振能量转移生物传感器还导致了Src和Rac相互调节的发现。我们的研究结果表明，同一信号反馈回路中的分子在不同的亚细胞位置可以受到不同的调节。总之，ECFP/YPet可作为一种通用的荧光共振能量转移对，用于开发高灵敏度的生物传感器，从而能够确定活细胞中亚细胞位置的分子层级。
Genetically encoded biosensors based on FRET have enabled the visualization of signaling events in live cells with high spatiotemporal resolution. However, the limited sensitivity of these biosensors has hindered their broad application in biological studies. We have paired enhanced CFP (ECFP) with YPet, a variant of YFP. This ECFP/YPet FRET pair markedly enhanced the sensitivity of biosensors (several folds enhancement without the need of tailored optimization for each individual biosensor) for a variety of signaling molecules, including tyrosine kinase Src, small GTPase Rac, calcium, and a membrane-bound matrix metalloproteinase MT1-MMP. The application of these improved biosensors revealed that the activations of Src and Rac by PDGF displayed distinct subcellular patterns during directional cell migration on micropatterned surface. The activity of Rac is highly polarized and concentrated at the leading edge, whereas Src activity is relatively uniform. These FRET biosensors also led to the discovery that Src and Rac mutually regulate each other. Our findings indicate that molecules within the same signaling feedback loop can be differentially regulated at different subcellular locations. In summary, ECFP/YPet may serve as a general FRET pair for the development of highly sensitive biosensors to allow the determination of molecular hierarchies at subcellular locations in live cells.