荧光相关光谱单分子检测用于转化生长因子受体激活的研究

项目名称： 荧光相关光谱单分子检测用于转化生长因子受体激活的研究

项目编号： No.91413119

项目类型： 重大研究计划

立项/批准年度： 2015

项目学科： 分析化学

项目作者： 方晓红

作者单位： 中国科学院化学研究所

项目金额： 50万元

中文摘要： 细胞信号转导的活细胞单分子研究是化学生物学交叉研究的前沿领域。本项目旨在发展细胞信号转导蛋白相互作用的原位、实时、动态、单分子研究新方法，将受激辐射耗尽（STED）超分辨成像技术引入到共聚焦荧光相关光谱技术中，在单分子水平上研究与恶性肿瘤发生发展密切相关的转化生长因子TGF-β信号转导中的重要分子事件，以深入了解TGF-β信号转导的分子机制。研究内容主要包括：(1) 搭建和优化基于STED超分辨荧光显微术的FCS系统, 使得探测焦斑体积更小、灵敏度更高; (2) STED-FCS应用于活细胞体系细胞膜上单分子扩散运动的表征, 通过探测不同条件下扩散系数等的改变研究转化生长因子受体的聚集和配体受体间的相互作用.该方法的建立将可推广至其它信号通路蛋白相互作用的研究.

中文关键词： 单分子荧光；超分辨显微镜；荧光相关光谱；信号转导；转化生长因子受体

英文摘要： Single-molecule fluorescence techniques has recently emerged as new and powerful tools to investigate the molecular mechanisms of cell signal transduction. This projects aims at developing new single-molecule methods for the real-time biomolecular interactions study of proteins involved in cell signaling. With the introduction of super-resolution stimulated emission depletion(STED) microscopic technique to fluorescence correlation spectroscopy (FCS), important molecular evens such as transforming growth factor-β (TGF-β) receptor activation will be studied at the single-molecule level in living cells. TGF-β and related growth factors regulate many important cellular processes, including the development and metastasis of cancer. We will firstly build the FCS system based on a STED microscope (STED-FCS) to achieve small detection volume and low background noise. Single-molecule diffusion monitoring will be realized on the membrane of living cells. The technique will be then applied to the study of TGF-β receptor oligomerization and ligand-receptor binding under different conditions. The results will offer new information for the understanding of TGF-β receptor activation. The method will also offer a new platform for the study of other cell signaling proteins.

英文关键词： single-molecule fluorescence；superresolutionmicroscope；fluorescence correlation spectroscopy；signaltransduction；transforming growth factor-β receptor