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的技术来可视化活细胞中的蛋白质单分子，以观察蛋白质之间的相互作用。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。检测表皮生长因子（EGF）、EGF受体（EGFR）单分子及相关信号分子。在本次资助期间，我们取得了以下成果：(1)细胞信号分子可视化：用绿色荧光蛋白（GFP）或其衍生物标记EGFR、小g蛋白Ras和细胞质丝氨酸/苏氨酸激酶c-Raf1，在不影响其生物活性的情况下，在活细胞中以单分子形式可视化。(2)检测EGF/EGFR复合物之间的分子相互作用：将Cy3-EGF和Cy5-EGF的混合物添加到表达EGFR和荧光共振烯的细胞中，在活细胞表面检测到从单个Cy3-EGF/EGFR复合物到另一个单个Cy5-EGF/EGFR复合物的更多能量转移（FRET）。观察到FRET效率的波动表明EGFR二聚体的结构波动。(3) egfr预聚类：用EGFR-GFP转染EGFR-GFP缺失细胞CHO-K1，观察细胞表面EGFR-GFP斑点荧光强度的变化。结果表明，egfr在加入EGF之前已形成预簇。人工修饰簇大小分布影响egf结合诱导的酪氨酸磷酸化。(4) Ras和Raf1之间的分子相互作用：在同时转染Ras和Raf1的细胞中，EGF刺激诱导Raf1从细胞质向质膜易位。从GFP-Raf1到YFP-Ras的FRET成像显示了Ras和Raf1的直接分子相互作用。相互作用在膜抽吸处延长超过60分钟。在单分子中可见的Raps和Raf1即使在膜上也能快速扩散，表明膜上活性分子的浓度是通过动态平衡来维持的。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