周质微丝在植物网格蛋白介导内吞中的功能研究

Clathrin-mediated endocytosis is the major endocytic portal of entry into cells, and plays a crucial role in signal transduction and the regulation of many plasma membrane activities. In plants, previously studies showed that auxin-activated ROP2 in the lobing region inhibits PIN1 endocytosis via the accumulation of cortical actin microfilaments induced by its effector RIC4. However, little is known about the molecular mechanism of the function of cortical actin in clathrin mediated endocytosis. In the present study, we will investigate the role of cortical actin in clathrin mediated endocytosis using living cell imaging techniques together with genetic approaches. First, we will monitor the dymamic colocalization of clathrin light chain (CLC) with cortical actin via time-lapse living cell imaging. Then, we will analyze the dynamics of CLC at the plasma membrane when the organization of cortical actin is changed. Finally, we will further explore in which steps cortical actin is involved in clathrin mediated endocytosis in plant cells. In the course of the investigation, some new methods and techniques including total internal reflection microscopy and fluorescence correlation microscopy will be used in this study to uncover the role of cortical actin in clathrin mediated endocytosis. The results of this study will shed light on the molecular mechanism of clathrin mediated endocytosis in plant developmental processes.

网格蛋白介导的内吞几乎存在于所有高等真核细胞中，是一种主要的内吞途径。 前人研究表明，生长素能够特异激活叶表皮细胞凸起 (lobe) 位置处的ROP2，并通过其下游作用元件RIC4促进周质微丝形成，从而抑制生长素转运蛋白PIN1的内吞，调控叶表皮细胞的发育。 然而，有关植物细胞中周质微丝调节网格蛋白介导的内吞的分子机制尚缺乏相关研究。本项目针对前人工作的不足，以拟南芥为材料，着重展开三个方面的研究：( 1) 研究周质微丝与网格蛋白包被囊泡的共定位关系 (2) 观察分析周质微丝改变条件下，网格蛋白 (CLC) 在细胞膜上的动态变化。(3) 探讨周质微丝调节网格蛋白介导的内吞的分子机理。在本项目研究中，拟采用新型可变角度全内反射荧光显微镜，荧光相关光谱等一些新方法、新技术，旨在揭示周质微丝在网格蛋白介导的内吞中的作用，对进一步揭示网格蛋白介导的内吞在植物生长发育中的分子机理具有重要的科学意义。