利用原子力显微镜单分子识别技术研究小窝蛋白在细胞膜上的分布

Caveolin is the major component of caveolae, which are cholesterol-rich vesicular invaginations of the plasma membrane. Caveolin involved in cellular cholesterol homeostasis, molecule transportation and transmembrane signaling events. The proposed project aims to locating the caveolin in quasi-native cell membranes by single-molecule recognition technology. Atomic Force Microscope (AFM) is well known as a very useful tool to observe single molecules due to its high-resolution and ability of imaging in variable environments. Especially, Topography and Recognition Imaging (TREC)，with several nanometers resolution has been recently developed to expand AFM’s application for specifically recognizing biological molecules. The project objectives are focused on the positioning and distribution characteristics of caveolin on the cell membrane. Determine the relationship between the cholesterol-rich region (lipid rafts) and caveolins. Furthermore, we will reveal the relationship between the caveolin in the membrane localization, distribution with carcinogenesis. Anticancer drugs also studied the role of change in tumor cell localization and distribution of caveolin. This study not only lay the foundation for the relationship between the structure and function of the cell membrane, But also to study the pathogenesis of cancer and the prevention, detection and treatment of cancer provides us with a new focus.

小窝蛋白是胞膜小窝结构的主要结构蛋白，能够与多种细胞信号分子相结合形成复合体，调控细胞关键信号分子的活性状态，进而调控细胞跨膜的信号转导参与细胞生命活动。本项目将致力于利用单分子识别成像技术对细胞膜上的小窝蛋白进行识别定位。基于原子力显微镜的单分子识别技术可以对细胞膜样品进行快速的高分辨识别扫描成像。本项目拟从单分子水平研究小窝蛋白在细胞膜上的定位及其分布特征。确定胆固醇富含区域（脂筏）与小窝蛋白的共定位关系。揭示小窝蛋白在细胞膜上的定位、分布与细胞癌变的关系。同时研究抗肿瘤药物作用于肿瘤细胞后小窝蛋白的定位及分布的变化。本研究不仅为在单分子水平研究细胞膜结构与功能的关系打下基础，而且还能够为我们研究癌症的发病机制以及癌症的预防、检测和治疗提供一个崭新的着眼点。

细胞膜上富含胆固醇区域与葡萄糖糖转运蛋白1蛋白簇具有明显的共定位关系。经环糊精处理破坏脂筏区域后，葡萄糖糖转运蛋白1的蛋白簇数量减少、簇的尺寸变小，说明脂筏在葡萄糖糖转运蛋白1聚集成簇中具有重要作用。确定了小窝与脂筏区域（富含胆固醇区域）的共定位关系。借助于葡萄糖糖转运蛋白的分布与癌症发生发展的关系，间接证明小窝蛋白在肿瘤细胞的分布特征与癌症的相关性。在纳米尺度下观察细胞膜上转运体的分布，为阐明这些功能蛋白调节因子以及获得分布与功能之间的关系提供了重要的信息。这为进一步研究葡萄糖转运蛋白，如转运体突变和癌症的发生发展之间的关系或者同时观察小窝蛋白富含区域与肿瘤的相互关联性提供了实验基础。