Development of the single-molecule technique to visualize protein-protein interactions in living cell and its application to the studies of cell signaling

这项研究的目的是开发新的技术来可视化活细胞中的蛋白质单分子，以观察蛋白质与蛋白质的相互作用。这些技术被应用于细胞信号分子反应的研究。利用全内反射荧光显微镜实现了单分子的可视化。(1)细胞信号分子的可视化：在不影响其生物学活性的情况下，用绿色荧光蛋白或其衍生物标记表皮生长因子、小G蛋白RAS和胞浆丝氨酸/苏氨酸激酶c-RAF1，并在活细胞中以单分子的形式显示它们。(2)检测表皮生长因子/表皮生长因子复合体之间的分子相互作用：在表达表皮生长因子和荧光共振烯…的细胞中加入细胞色素3-表皮生长因子和5-表皮生长因子的混合物在活细胞表面检测到从单一的Cy3-EGF/EGFR复合体到另一个单一的Cy5-EGF/EGFR复合体的Rgy转移(FRET)。观察到FRET效率的波动，提示EGFR二聚体的结构变化。(3)EGFR的预聚簇：将EGFR-GFP导入EGFR阴性细胞(CHO-K1)，检测细胞表面EGFR-GFP斑点的荧光强度变化。结果表明，在加入EGF之前，EGFR形成了预团簇。(4)RAS和Raf1之间的分子相互作用：在同时转导RAS和Raf1的细胞中，EGF刺激诱导Raf1从胞浆转位到质膜。从GFP-Raf1到YFP-RAS的FRET成像显示Ras和Raf1的直接分子相互作用。这种相互作用在膜抽签处延长了60min以上。单分子中可见的RAPs和Raf1都在快速扩散，甚至在膜抽奖中也是如此，这表明抽奖中活性分子的浓度保持了动态平衡。较少

The aim of this study was the development of new techniques to visualize single molecules of proteins in living cells to observe protein-protein interactions. The techniques were applied to the studies of the reactions of cell signaling molecules. Visualization of single molecules was achieved by using total internal reflection fluorescence microscopy. Single molecules of epidermal growth factor (EGF), EGF receptor (EGFR) and related signaling molecules were detected.During the period of this grant, we obtained the following achievements.(1) Visualization of cell signaling molecules: EGFR, small G-protein Ras and cytoplasmic serine/threonine kinase c-Raf1 were tagged with green fluorescent protein (GFP) or its derivatives without affecting their biological activities and were visualized as single molecules in living cells.(2) Detection of molecular interactions between EGF/EGFR complexes: a mixture of Cy3-EGF and Cy5-EGF was added to cells expressing EGFR and fluorescence resonance ene … More rgy transfer (FRET) from single Cy3-EGF/EGFR complex to another single Cy5-EGF/EGFR complex was detected on a living cell surface. Fluctuation of the FRET efficiency was observed suggesting structural fluctuation of EGFR dimers.(3) Pre-clustering of EGFRs: an EGFR-null cell (CHO-K1) was transfected with EGFR-GFP and variation of the fluorescence intensity of EGFR-GFP spot on the cell surface was assessed. The result indicated that EGFRs formed pre-clusters before addition of EGF. Artificial modification of the cluster size distribution affected tyrosine phosphorylation induced by EGF-binding.(4) Molecular interactions between Ras and Raf1: in cells transfected with both Ras and Raf1, stimulation with EGF induced translocation of Raf1 from the cytoplasm to the plasma membrane. Direct molecular interactions of Ras and Raf1 were visualized by FRET imaging from GFP-Raf1 to YFP-Ras. The interactions were prolonged at the membrane raffles for more than 60 min. Both Raps and Raf1 visualized in single molecules were rapidly diffusing even in the membrane raffles indicating the concentration of active molecules in the raffles was maintained by dynamic equilibrium. Less