TIRFM-FCS成像系统搭建及其在膜蛋白单分子应用的研究

在生物活细胞中，膜蛋白分子动态行为的实时无损检测与分析，是生命科学研究中迫切需要解决的问题。然而细胞膜的纳米尺度结构及其分子事件的微秒级时间分辨率，却限制了对其分子行为的可视化追踪成像。为此有必要发展超时空分辨率的成像技术,从而在单分子尺度上探测细胞生命活动的单个、相对独立的早期事件。本项目拟在我们过去研究工作的基础上，进一步创研出一种基于全内反射荧光显微术（TIRFM）和荧光相关光谱（FCS）相结合的实时高分辨率荧光成像与定量分析技术。并以植物细胞膜上G蛋白偶联受体GTG1、GTG2为研究对象，对它们的侧向运动、寡聚化和内吞进行活体动态进行分析。以实现植物细胞膜蛋白对环境刺激响应早期事件中时空动态分析及可视化成像，旨在拓展单分子检测技术的综合联用，从而为膜蛋白活性调节这一重要生物学问题的研究提供关键的技术手段。

The dynamic behavior of membrane proteins in living cells in real time non-destructive testing and analysis is an urgent demand for life science research. However, the nano-scale spatial resolution of the membrane and the molecular events microsecond time resolution limited the visualization of the behaviors of these molecules. Thus, a super dimension resolution of the imaging techniques need to be developed to investigate the relative independent and early events of life activities in the single-molecule levels. Based on our earlier investigations, considered to the technical insufficiency to study membrane porteins dynamics in living cells, we will develop a real-time and high-resolution fluorescence quantitative analysis system. Applying this TIRFM-FCS platform, we will carry out experiments to study the lateral movement, oligomerization, and endocytosis of plant membrane G protein-coupled receptor GTG1 and GTG2, to clarify the membrane protein early response to environmental stimuli in space-time dynamics and their visualization. The present project will expand the single-molecule detection technology associated with the key techniques for investigating the activity of membrane proteins and their regulations in this important biological process.