囊泡转运参与重要膜蛋白功能的调控机制

As main components of plasma membrane in plant cells, membrane proteins play a major role in several biological processes. It is well demonstrated that the biphasic transport mediated by vesicle trafficking is crucial for macromolecule transportation and signaling between the cells and their external environment. However, the mechanism of how vesicle trafficking regulates membrane proteins to maintain their homeostasis in plants remains to be further elucidated. In consideration of the insufficiency of previous investigations, this project mainly focuses on the endocytic pathways and intracellular trafficking routes by means of dynamic analysis in the living cells together with genetic approaches in 6 different membrane proteins. First, we will determine the distribution patterns and dynamic properties of membrane proteins, as well as their oligomerization states in different membrane microdomains. Then we will compare the endocytic pathways and endocytic velocities in 6 different membrane proteins. Moreover, we are going to investigate the intracellular trafficking and final destination of membrane proteins. Finally, we will further explore the variations in dynamic properties, endocytic pathways and intracellular trafficking routes of membrane proteins in response to different treatments. In this investigation, some new techniques including variable-angle total internal reflection fluorescence microscopy, fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy will be used. Surely, the results of this investigation will lay foundation for the better understanding of the regulatory mechanisms of vesicle trafficking involved in the functional characterization of membrane proteins in plants.

植物细胞膜蛋白作为细胞膜的重要组分，是许多生物学功能的执行者。由囊泡转运所介导的双向性运输在细胞内外物质交换和信号传递过程中发挥重要作用。然而，囊泡转运是如何调控细胞膜蛋白的功能，在植物中尚缺乏深入的研究。针对前人工作的不足，本项目将以拟南芥中两类6个膜蛋白为研究对象，着重开展四方面的研究：(1)分析膜蛋白在质膜上的运动和聚合状态，以及在膜微区内外的分布与运动规律；(2) 比较分析膜蛋白的胞吞方式及胞吞速率；(3) 膜蛋白的胞内转运途径和蛋白去向的研究；(4) 不同处理后，膜蛋白的运动特性、胞吞方式和胞内转运变化规律的研究。在本项目中，拟采用可变角度的全内反射荧光显微术、荧光相关光谱、荧光互相关光谱等新技术。无疑，本研究结果将为阐释植物细胞中囊泡转运参与膜蛋白功能的调控机制奠定基础。

膜蛋白是生物膜功能的主要承担者，参与多种外界信息交流等生命活动。膜蛋白在感知外界信号以后，会在细胞膜上发生快速侧向位移、聚合，进而以膜泡的形式进行胞吞。膜泡运输系统除了参与物质吸收等基本生理过程外，也在膜蛋白功能的调控中发挥重要作用。尽管已有报道对多种膜蛋白的生理功能等进行了研究，然而囊泡转运如果调控蛋白功能尚不明晰。本项目发展和完善了适合在植物活体中，开展无损伤、高时空分辨率成像和分析的技术。应用这些技术和方法，本项目主要开展了如下研究：(1) 分析phot1在细胞质膜上感知蓝光前后发生的动态变化，揭示膜微区在phot1介导的蓝光信号传递过程中所扮演的角色；(2) 系统性的对FER-GFP胞吞及其转运过程的分析，揭示了RALF1-FER互作促进多种细胞膜蛋白的胞吞是RALF1抑制拟南芥根伸长的重要细胞学机制；(3) 阐明了膜脂重要组分——固醇参与被激活的类受体激酶的胞吞，进而在植物对细菌的早期免疫防御反应中起重要作用；(4) 探讨了PEPR1蛋白的分布、运动、聚合和胞吞之间的联系，并揭示了膜筏参与调控PEPR1的动态和胞吞。综上所述，本项目通过在活体植物材料中对膜蛋白和膜泡运输的动态进行高时空分辨率的解析，阐释了膜泡运输调控膜蛋白活性，进而参与植物对外界信号的早期应答的细胞学机制。