网格蛋白是由重链CHC和轻链CLC组成的复合体，其介导的内吞是动植物细胞最重要的内吞途径之一，直接调控胞外物质吸收、质膜蛋白丰度和胞内外信号传导等重要细胞生物学过程。前期研究表明，拟南芥CLC2和CLC3功能缺失影响生长素极性输出载体PIN2内吞与根尖向地性，但不影响其极性定位，而CLC1功能缺失引起花粉致死。因此轻链细胞生物学功能一直没有最后定论。本申请项目利用根尖特异性表达启动子和反义RNA抑制技术，成功制备根尖轻链功能缺失三突变体。在此基础上，拟开展以下几项研究工作：（1）分析轻链功能缺失对PIN2极性定位、内吞与回收、根尖向地性与生长素分布的影响；（2）分析轻链在重链质膜招募与装配中的调控功能；（3）分析轻链在网格蛋白接头蛋白亚基质膜招募中的作用。通过这些研究期望能深层次剖析轻链调控内吞的分子机理及其在PIN极性定位中的生物学功能，为研究PIN介导生长素极性运输机制提供新的见解。

Clathrin is composed of clathrin heavy chains (CHC) and light chains (CLC). Clathrin-mediated endocytosis (CME), a major endocytic pathway in animal and plant cells, directly regulates many cellular processes including the uptake of extracellular nutrients, abundance of plasma membrane (PM) proteins, and transduction between extra- and intracellular signals. Previous studies have shown that loss of CLC2 and CLC3 impairs endocytosis of PIN2 auxin efflux transporter and root gravitropism but does not affect polar localization of PIN2. Moreover, loss of CLC1 causes a pollen lethality phenotype, which significantly hinders us from genetically characterizing the cellular functions of CLCs. In this study, we used the root tip-specific promoter PIN2 and antisense RNA technique and successfully generated loss-of-function clc triple mutants. Based on these results, we will focus on the following key points: (1) analyze the effects of loss of CLCs on the PM polar localization, endocytosis and recycling from/to the PM of PIN2, root gravitropism and asymmetric auxin distribution; (2) identify function roles of CLCs in the recruitment of CHCs to the PM; (3) characterize function roles of CLCs in the recruitment of clathrin adaptor proteins AP2 (adaptor protein 2) and TPC (TPLATE complex) to the PM. These analyses would deeply dissect molecular mechanisms underlying CLC regulation of endocytosis and their functional roles in PIN polar localization, which will provide new insights into the regulation mechanisms of PIN-mediated polar auxin transport in plants.