拟南芥硼转运体NIP5;1和BOR1质膜分子动态及其内吞机制研究

Endocytosis plays important roles in extracellular substance intake and intracellular protein transportation in plant growth and development, while microdomain-resided proteins are crucial factors regulating endocytic activity and intracellular transportation. However, protein dynamics in certain membrane microdomains and corresponding endocytic machinery are still largely uncovered. Boron transportation in plant cells are dependent on boron transporters showing typical polarized distribution, namely NIP5;1 and BOR1. NIP5;1 and BOR1 interact with endocytic components in plasma membrane in response to variations in extracellular boron concentration, which makes them ideal targets for investigating endocytic pathway and transportation machinery. The present study will be carried out on Arabidopsis seedlings using single particle tracking and other techniques with reference to the shortage in previous studies. Firstly, we will determine the distribution patterns of NIP5;1 and BOR1 on plasma membrane before and after induction, kinetic parameters will be included for further analysis; secondly, colocalization analysis will be performed to examine the relationship of NIP5;1 and BOR1 to previously identified endocytic components clathrin and flotillin; the endocytic pathway and intracellular transportation routes of NIP5;1 and BOR1 following internalization will be further investigated. The results will illustrate the mechanism for specific selective modulation on the balance in boron transportation by receptor-mediated endocytosis, and provide further evidence for spatio-temporal molecular machinery for receptor-mediated endocytosis and signaling initiation.

细胞内吞参与维持胞外物质吸收和胞内蛋白转运，而质膜微区蛋白是调节胞吞活性和胞内转运的关键因子。植物细胞中硼的吸收和转运依赖于极性分布的硼转运体NIP5;1和 BOR1。高硼诱导条件下NIP5;1和 BOR1在细胞质膜与内吞组件相互作用，是研究植物细胞内吞作用和硼转运机制的理想体系。本项目针对前人工作不足，以模式植物拟南芥为材料，运用单颗粒追踪、荧光相关光谱等技术，着重开展三方面的研究：(1) 确定硼转运体NIP5;1和 BOR1在外源硼诱导前后的质膜定位模式，对比分析其动力学特征；(2) 研究NIP5;1和 BOR1与内吞途径标志蛋白在质膜上的互作和内吞平衡机制；(3) 分析NIP5;1和 BOR1经膜微区的内吞及胞内转运途径。相关研究结果将有助于阐明受体介导的内吞选择性调节硼转运动态平衡的机理，为探讨内吞途径调控质膜受体蛋白及其信号激发的时空特异性分子机制提供依据。

细胞内吞参与维持胞外物质吸收和胞内蛋白转运，而质膜微区蛋白是调节胞吞活性和胞内转运的关键因子。植物细胞中硼的吸收和转运依赖于极性分布的硼转运体BOR1和DUR3。高硼诱导条件下BOR1和DUR3在细胞质膜与内吞组件相互作用，是研究植物细胞内吞作用和硼转运机制的理想体系。本项目针对前人工作不足，以模式植物拟南芥为材料，运用单颗粒追踪、荧光相关光谱等技术，着重开展如下研究：(1) 通过遗传转化，获得了bor1-1突变体背景下，稳定表达pBOR1::BOR1-eGFP的拟南芥株系；(2) 利用激光共聚焦显微镜和全内反射荧光显微镜对pBOR1::BOR1-eGFP在细胞质膜上的表达和动力学参数进行了观测；(3) 筛选获得了作为寡聚化分析中单体GFP对照的myristoyl-mGFPA206K转基因拟南芥株系；(4) 完善并更新VAEM成像平台的数据采集参数及数据分析方法；(5) 利用更新后数据采集参数及数据分析方法对硼转运体BOR1在质膜近膜区的单分子动态分析；(6) 硼转运体DUR3在质膜近膜区的单分子动态分析。相关研究结果将有助于阐明受体介导的内吞选择性调节硼转运动态平衡的机理，为探讨内吞途径调控质膜受体蛋白及其信号激发的时空特异性分子机制提供依据。