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

本研究的目的是开发新技术，以可视化活细胞中的蛋白质单分子，观察蛋白质-蛋白质相互作用。该技术已应用于细胞信号分子反应的研究。单分子的可视化是通过使用全内反射荧光显微镜实现的。表皮生长因子（EGF）、表皮生长因子受体（EGFR）及相关信号分子的单分子检测。(1)细胞信号分子的可视化：EGFR、小G蛋白Ras和细胞质丝氨酸/苏氨酸激酶c-Raf 1用绿色荧光蛋白（GFP）或其衍生物标记，而不影响其生物活性，并在活细胞中可视化为单分子。(2)检测EGF/EGFR复合物之间的分子相互作用：将Cy 3-EGF和Cy 5-EGF的混合物加入表达EGFR的细胞中，荧光共振成像检测EGF/EGFR复合物之间的分子相互作用。 ...更多信息 在活细胞表面上检测到从单个Cy 3-EGF/EGFR复合物到另一个单个Cy 5-EGF/EGFR复合物的荧光共振能量转移（FRET）。观察到FRET效率的波动，表明EGFR二聚体的结构波动。(3)EGFR的预聚类：用EGFR-GFP转染无EGFR细胞（CHO-K1），并评估细胞表面上EGFR-GFP斑点的荧光强度的变化。结果表明，在加入EGF之前，EGFRs形成了预聚体。人工修饰的簇大小分布影响EGF结合诱导的酪氨酸磷酸化。(4)Ras和Raf 1之间的分子相互作用：在同时转染Ras和Raf 1的细胞中，EGF刺激诱导Raf 1从细胞质易位到质膜。Ras和Raf 1的直接分子相互作用通过从GFP-Raf 1到YFP-Ras的FRET成像可视化。的相互作用被延长在膜抽气超过60分钟。这两个RAP和Raf 1可视化在单分子迅速扩散，即使在膜抽气表明活性分子的浓度在抽气中保持动态平衡。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