纤毛组装与运动中关键超大分子复合体IFT及RS的冷冻电镜结构研究

Cilia and flagella are highly conserved organelles that play important roles in the movement, perception and signal transduction in different eukaryotic species. Ciliary dysfunction results in numerous human diseases, including infertility, polycystic kidney, etc. Macromolecular complexes such as intraflagellar transport (IFT) complex and radial spokes (RS) complex serve pivotal roles in cilia assembly and movement. IFT complex consists of 21 distinct proteins, and it can move bidirectionally underneath the ciliary or flagellar membrane between the basal body and the distal tip of the axoneme. It is responsible for cilia maintenance, assembly and growth. RS complex, formed by at least 23 different proteins, is located between outer microtubule doublets and 2 single central microtubules, influencing ciliary movement and signaling. To date, only a few low resolution (~40 Å) in situ electron-tomography (ET) structures are available for each of the two complexes with the sample in plastic-embedding condition. Due to the complexity of the two macromolecules, purification as well as high-resolution structure determination by classical biophysical methods remain challenging. As a result, lacking of high-resolution structural information hinders our understanding of the underlying molecular mechanism of their assembly and function in cilia movement. The goal of our research is to resolve high-resolution cryo-EM structures of the key sub-complexes of IFT and RS complexes utilizing cutting edge cryo-EM technique, and to uncover the architecture of the machinery as well as their structure-function relationship in cilia assembly and movement. This will facilitate developments in the diagnosis and treatment of related ciliary human diseases.

纤毛和鞭毛是真核生物中普遍存在并高度保守的细胞器，在不同真核生物的运动、感知和信号传递中发挥重要作用。纤毛的缺欠通常会导致多种人类疾病，包括不育、多囊肾等。鞭毛运输（intraflagellar transport，IFT）及辐射轴（Radial spoke，RS）是纤毛中重要的超大分子复合体。IFT由21种蛋白质组成，密切参与纤毛内物质运输，在纤毛的组装及维持中发挥重要作用。RS由至少23种蛋白质组成，位于纤毛中心微管和周围微管二联体之间，影响纤毛的运动和信号传递。目前IFT和RS复合体的结构信息仅限于低分辨率（~40Å）的原位电子断层三维重构，高分辨率结构信息的缺失制约了人们对其形貌构架及其行使功能的分子机理的认知。本项目拟应用冷冻电镜技术解析超大分子复合体IFT及RS的关键亚复合体高分辨率三维结构，阐释其构架组成及其参与纤毛组装与运动的分子机理，进而为纤毛相关疾病的诊疗提供理论基础。

纤毛是广泛存在于真核生物中，且高度保守的细胞器，在生物的运动、感知和信号传递中发挥重要作用。纤毛的异常会导致包括不育、多囊肾在内的多种人类疾病。纤毛中重要的超大分子复合体包括鞭毛运输（intraflagellar transport，IFT）及辐射轴（Radial spoke，RS）。IFT由至少22种蛋白质组成，参与纤毛内货物运输，对维持纤毛内稳态有着重要作用。RS由至少23种蛋白质组成，位于纤毛中心微管和周围微管二联体之间，参与纤毛的运动和信号传递。本项目应用冷冻电镜技术对哺乳动物超大分子复合体IFT及RS的关键亚复合体进行了结构解析。本项目资助的主要研究进展如下：1）首次解析了哺乳动物辐射轴头部（RS head）复合体3.2 Å高分辨率的冷冻电镜结构并从头建模，此为首个高等动物RS头部复合体的原子结构，揭示了各亚基的作用网络，对纤毛疾病的关键突变位点进行了定位，提出了辐射轴-中央微管（RS-CP）相互作用的模型和机制（Proc Natl Acad Sci, 2021）。2）纯化了IFT-A核心复合体，并进行了初步结构解析。3）已完成哺乳动物IFT-B超大复合体所有亚基的表达，并对核心复合体IFT-B1 core-2进行了纯化及结构解析。在Proc Natl Acad Sci, Molecular Cell, Nat Commun等高水平期刊发表论文3篇。