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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Determination Of Hierarchical Relationship Of Src And Rac At Subcellular Locations With Fret Biosensors
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Determination Of Hierarchical Relationship Of Src And Rac At Subcellular Locations With Fret Biosensors

机译：用Fret生物传感器确定亚细胞位置Src和Rac的等级关系

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Genetically encoded biosensors based on FRET have enabled the visualization of signaling events in live cells with high spatiotem-poral 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.
机译：基于FRET的基因编码生物传感器使可视化具有高时空孔分辨率的活细胞中的信号事件。然而，这些生物传感器的有限的灵敏度阻碍了它们在生物学研究中的广泛应用。我们已将增强型CFP（ECFP）与YPet（YFP的变体）配对。这种ECFP / YPet FRET对显着增强了生物传感器对多种信号分子（包括酪氨酸激酶Src，小GTPase Rac，钙和膜结合蛋白）的敏感性（无需针对每个生物传感器进行量身定制的优化，可以提高几倍）。基质金属蛋白酶MT1-MMP。这些改进的生物传感器的应用表明，PDGF对Src和Rac的激活在微图案表面的定向细胞迁移过程中显示出不同的亚细胞模式。 Rac的活性高度极化并集中在前缘，而Src活性相对均匀。这些FRET生物传感器还导致发现Src和Rac相互调节。我们的发现表明，同一信号反馈环内的分子可以在不同的亚细胞位置被不同地调节。总之，ECFP / YPet可以用作通用FRET对，用于开发高度敏感的生物传感器，从而可以确定活细胞中亚细胞位置的分子层次。

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