植物细胞网格蛋白介导的内吞与外吐的稳态调控机理研究

Clalthrin-mediated endocytosis (CME) and exocytosis, a major mode for membrane protein trafficking in eukaryotic cells, directly regulate many important cellular processes including secretion and uptake of intra- and extracellular materials, abundance of important plasma membrane (PM) proteins, and transduction of intra- and extracellular signals. It is well known that endocytosis and exocytosis are two tightly coupling regulation processes, which is critical for cell division, differentiation, and growth. In plant cells, clathrin predominantly localizes at the PM and trans-Golgi network/early endosome (TGN/EE). Clathrin recruitment to the PM is synergistically mediated by PM-localized adaptor protein 2 (AP2) and TPLATE complex (TPC), whereas its recruitment to the TGN/EE is mediated by TGN/EE-localized AP1 complex. However, whether a coupling regulation mechanism between CME and exocytosis operates in plant cells is largely unknown so far. This proposal will use genetic and pharmacological strategies to selectively interfere with Arabidopsis AP2/TPC-mediated endocytosis and AP1-mediated exocytosis and consequently reveal a coupling regulation mechanism underlying CME from the PM and exocytosis from the TGN/EE in plant cells. These studies will primarily elucidate the coupling regulation mechanism between CME and exocytosis in plants, which provides a novel mechanism for the interaction network of the PM and intracellular organelles in eukaryotic cells and also provide new insights into key scientific issues of this major research project.

网格蛋白介导的内吞与外吐是真核细胞膜蛋白运输的最主要方式之一，直接调控胞内外物质的分泌与吸收、重要质膜蛋白的丰度和胞内外信号的传递等重要细胞生物学过程。动物细胞质膜内吞与外吐是两个紧密偶联调控的过程，对细胞分裂分化与生长具有重要生物学意义。植物细胞网格蛋白主要定位于质膜和反式高尔基网络结构/早期内体(TGN/EE)，其招募分别由质膜定位的接头蛋白AP2/TPC和TGN/EE定位的AP1介导完成，但至今仍不清楚植物细胞质膜内吞与外吐之间是否存在互作调控机制。本申请项目运用遗传学和药理学方法选择性地干涉拟南芥AP2/TPC介导的内吞和AP1介导的外吐途径从而剖析植物细胞质膜内吞与TGN/EE外吐之间的互作调控机理，初步阐明植物网格蛋白介导的内吞与外吐途径之间的互作调控机理，为解析真核细胞质膜与胞内细胞器之间的互作网络提供新的互作机制，为本重大研究计划的核心科学问题提供新的观点与见解。

网格蛋白介导的内吞与外吐是胞内外物质的吸收和分泌、重要质膜蛋白丰度调控和胞内外细胞信号传递的重要途径，是真核细胞生长发育的重要调控机制之一。虽然动物细胞中内吞与外吐之间的紧密偶联调控已较为清楚，但植物细胞内吞与外吐是否存在调控关系仍缺乏证据。因此，深入开展研究植物网格蛋白介导的内吞与外吐之间的分子调控机理研究具有重要的科学意义。在本项目的资助下，目前已获得以下主要研究结果：（1）初步阐明了AP-1介导的外吐对网格蛋白介导的内吞的影响。活细胞成像分析表明，AP-1功能缺失抑制质膜蛋白PIN2的内吞和内吞示踪染料FM4-64的吸收，为进一步剖析外吐调控网格蛋白介导的内吞过程提供了细胞学证据；（2）分析了AP-1功能破坏对网格蛋白、接头蛋白AP-2和TPC质膜招募的影响。利用激光共聚焦显微镜、免疫定位和TIRF活细胞成像技术分析结果显示，AP-1亚基敲除导致网格蛋白、AP-2和TPC质膜招募受抑制，从而在分子水平上剖析了AP-1介导的外吐如何调控网格蛋白介导的内吞的分子机理；（3）破坏网格蛋白介导的内吞，质膜蛋白PIN2的质膜重回收被抑制。同时，分泌蛋白标记Sec-GFP在胞内信号积累显著增加。这些分析结果表明，网格蛋白介导的内吞参与调控质膜蛋白重回收和分泌过程；（4）活细胞成像和Western blotting分析表明，ap-2和tpc突变体中AP-1在TGN/EE的招募被抑制，初步阐明网格蛋白介导的内吞调控外吐的分子机理。以上实验结果证明植物细胞中网格蛋白介导的内吞和外吐途径之间存在协同调控现象，对进一步理解网格蛋白介导的PIN内吞与外吐在生长素极性运输的分子机理具有重要的科学意义，为深入研究真核生物细胞质膜与胞内细胞器之间的互作网络提供新的互作机制。.受本项目资助下，已发表学术论文SCI刊物3篇，另有1篇Plant cell稿件已修回。培养研究生8人，正在培养19人。国际或全国性学术会议作报告1次。