Spying on protein interactions in living cells with reconstituted scarlet light.
Spying on protein interactions in living cells with reconstituted scarlet light.
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DOI：
10.1039/c8an01223g
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发表时间：
2018-10
期刊：
影响因子：
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通讯作者：
Sheng Wang;M. Ding;Boxin Xue;Yingping Hou;Yujie Sun
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文献类型：
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作者：
Sheng Wang;M. Ding;Boxin Xue;Yingping Hou;Yujie Sun
双分子荧光互补（BiFC）测定和BiFC联合荧光共振能量转移（FRET）技术已成为研究活细胞分子相互作用的重要工具。然而，大多数现有的红色荧光蛋白衍生的BiFC检测方法的真实检测和细胞成像性能仍然存在着集合亮度相对较低、细胞毒性高、红色荧光蛋白在活的哺乳动物细胞中存在早聚集或严重的残留二聚体、互补效率低、37℃生理温度下成熟缓慢等问题。我们开发了一种基于最近发展的真正单体红色荧光蛋白（FP） mScarlet-I的BiFC检测，该蛋白具有优异的细胞性能，如低细胞毒性，快速有效的发色团成熟以及在所有先前报道的单体红色荧光蛋白中最高的细胞内亮度。本研究采用经典的β-Fos/β-Jun本构异质二聚化模型和雷帕霉素诱导的FRB/FKBP相互作用系统，建立并测试了基于mscarlett -i的活哺乳动物细胞BiFC检测的性能。此外，只需采用大斯托克斯位移荧光蛋白mAmetrine作为供体，通过将活细胞敏化发射FRET测量与基于mscarlet - i的BiFC检测相结合，就可以在活HeLa细胞中方便有效地检测和可视化β-Jun-β-Fos-NFAT1三联蛋白复合物的形成，且光谱串导最小。目前在这项工作中建立的BiFC分析也被证明能够在37°C生理温度下在活的哺乳动物细胞中以高特异性和灵敏度检测和可视化不同亚细胞区室的各种蛋白质-蛋白质相互作用（PPIs）。
The BiFC (bimolecular fluorescence complementation) assay and BiFC combined with FRET (fluorescence resonance energy transfer) technique have become important tools for molecular interaction studies in live cells. However, the real detection and cellular imaging performances of most existing red fluorescent protein-derived BiFC assays still suffer from relatively low ensemble brightness, high cytotoxicity, the red fluorescent proteins being prone-to-aggregation or severe residual dimerization, inefficient complementation and slow maturation at 37 °C physiological temperature in live mammalian cells. We developed a BiFC assay based on a recently evolved truly monomeric red fluorescent protein (FP) mScarlet-I with excellent cellular performances such as low cytotoxicity, fast and efficient chromophore maturation and the highest in-cell brightness among all previously reported monomeric red fluorescent proteins. In this work, a classic β-Fos/β-Jun constitutive heterodimerization model and a rapamycin-inducible FRB/FKBP interaction system were used to establish and test the performance of the mScarlet-I-based BiFC assay in live mammalian cells. Furthermore, simply by adopting the large-Stokes-shift fluorescent protein mAmetrine as the donor, β-Jun-β-Fos-NFAT1 ternary protein complex formation could be readily and efficiently detected and visualized with minimal spectral cross-talk in live HeLa cells by combining live-cell sensitized-emission FRET measurement with the mScarlet-I-based BiFC assay. The currently established BiFC assay in this work was also shown to be able to detect and visualize various protein-protein interactions (PPIs) at different subcellular compartments with high specificity and sensitivity at 37 °C physiological temperature in live mammalian cells.