Structural and functional studies of axonemal microtubule inner proteins (MIPs)

轴丝微管内部蛋白（MIP）的结构和功能研究

• 批准号: 10448243
• 负责人: Rui Zhang
• 金额: $ 36.86万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-10 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10448243
• 关键词: Architecture; Basic Science; Beak; Biological Process; C-terminal; Cell physiology; Charge; Chlamydomonas; Chronic; Cilia; Cryoelectron Microscopy; Data; Dependence; Etiology; Eukaryota; Eukaryotic Cell; Filament; Flagella; Functional disorder; Genetic; Hair; Handedness; Hearing; Human; Impairment; Individual; Infertility; Length; Location; Microtubule-Associated Proteins; Microtubules; Molecular; Organelles; Organism; Orthologous Gene; Perception; Periodicity; Positioning Attribute; Proteins; Radial; Registries; Research; Resolution; Respiratory Tract Infections; Running; Sensory; Shapes; Site; Smell Perception; Structure; Surface; Tail; Testing; Tubulin; Vision; Work; base; cell motility; ciliopathy; cilium biogenesis; cilium motility; fluid flow; human disease; in vitro Assay; in vivo; mutant; nanomachine; particle; protein complex; research study; scaffold; tektins

Cilia (also known as flagella) are hair-like organelles protrude from the surface of most eukaryotic cells and are responsible for cellular motility, fluid flow and sensory perception. A large group of human diseases, known as ciliopathies, are caused by cilia dysfunction. The elongated shape of the cilium is supported by a highly conserved structure called the axoneme. In most motile cilia, the axoneme has a 9+2 architecture in which nine doublet microtubules (DMTs) surround a central pair of singlet microtubules (MTs). Bound periodically along the length of each DMT are a variety of MT-associated proteins and complexes that decorate the external and luminal surfaces with different periodicities (8,16, 24, 48 and 96-nm). These protein complexes are found in coherent register along the entire length of the DMT, and loss of the coherence causes impaired motility. How periodicity is established, maintained, and synchronized, especially over a long distance, has been a long-standing question in the field. Each DMT has a distinctive structure with one complete ring of A- tubule and one incomplete ring of B-tubule. How the unique architecture of the DMT is formed in vivo is still unclear. Furthermore, many axonemal components are asymmetrically distributed in both the longitudinal direction and the radial direction among the 9 DMTs. For example, 3 of the 9 DMTs contain a unique “beak” structure in the proximal B-tubule lumens. To date, the molecular components and biological functions of the beak are unknown. In this proposal, based on the identification of 33 microtubule inner proteins (MIPs) in our recent work using high-resolution cryo-electron microscopy (cryo-EM), we propose to elucidate the functions of individual MIPs during ciliogenesis using Chlamydomonas mutants. The objective of this application is to use a combination of genetic and structural approaches to investigate the architectural principles governing the assembly of DMTs and axonemes. We will focus on three key aspects of the architectural principles with the following specific aims: (1) We will identify the key proteins responsible for maintaining coherent registry between different periodicities, and investigate their mutual dependence, using Chlamydomonas mutants lacking filamentous MIPs, external coiled-coil proteins, and proteins located at interfaces between different repeat regions. (2) We will investigate the molecular mechanism governing B-tubule formation, and test two hypotheses: (i) proteins located at the MT seam, the unique site within the A-tubule, are essential for B-tubule formation; (ii) MIPs located at the outer junction (OJ) function to promote the B-tubule formation by shielding the inhibitory effects of tubulin C-terminal tails at the OJ. (3): We will identify protein components of the beak using high-resolution cryo-EM. Our preliminary data suggest that two main components are tektin filaments and SAXO proteins. We will investigate their cellular functions and relevance to human diseases using Chlamydomonas mutants and in vitro assays. Most of the proteins studied here have orthologs in human cilia; therefore, our work will provide a molecular basis for understanding the etiology of many human ciliopathies.

纤毛（也称为鞭毛）是从大多数真核细胞表面突出的毛发状细胞器， 负责细胞运动、液体流动和感觉知觉。一大类人类疾病， 纤毛病是由纤毛功能障碍引起的。纤毛的细长形状是由一个高度 称为轴丝的保守结构。在大多数运动纤毛中，轴丝具有9+2结构，其中 九个双峰微管（DMT）围绕中心的一对单峰微管（MT）。周期性束缚 沿着每个DMT的长度是多种MT相关蛋白和复合物，其装饰DMT。 具有不同周期（8、16、24、48和96-nm）的外表面和腔表面。这些蛋白质复合物 沿着DMT的整个长度沿着在相干寄存器中发现，并且相干性的损失导致受损 能动性周期性是如何建立、维持和同步的，特别是在长距离上， 这是该领域长期存在的问题。每个DMT具有独特的结构，具有一个完整的A-环， 小管和一个不完整的B-小管环。DMT的独特结构如何在体内形成仍然是未知的。 不清楚此外，许多轴丝成分在纵向和纵向上都是不对称分布的。 方向和径向方向之间的9个DMT。例如，9种DMT中有3种含有独特的“喙”。 近端B小管管腔中的结构。迄今为止，对其分子组成和生物学功能进行了研究 鸟嘴未知。在此基础上，我们鉴定了33种微管内蛋白（MIPs）， 最近的工作，使用高分辨率冷冻电子显微镜（cryo-EM），我们建议阐明的功能， 在纤毛发生过程中使用衣原体突变体的单个MIP。此应用程序的目标是使用 遗传学和结构学方法相结合，以研究管理 DMT和轴丝的组装。我们将重点讨论架构原则的三个关键方面， 具体目标如下：（1）我们将确定负责维持一致性登记的关键蛋白质 之间的不同周期性，并调查他们的相互依赖性，使用衣原体突变体 缺乏丝状MIP、外部卷曲螺旋蛋白和位于不同蛋白质之间界面的蛋白质， 重复区域。(2)我们将研究控制B-小管形成的分子机制，并测试两个 假设：（i）位于MT接缝（A-小管内的独特位点）的蛋白质对于B-小管是必不可少的。 （ii）位于外连接（OJ）的MIP通过屏蔽作用促进B-小管的形成 微管蛋白C末端尾在OJ处的抑制作用。(3)：我们将鉴定喙的蛋白质成分 使用高分辨率冷冻电镜我们的初步数据表明，两个主要组成部分是tektin细丝 SAXO蛋白质我们将研究他们的细胞功能和人类疾病的相关性， 衣原体突变体和体外试验。这里研究的大多数蛋白质在人类纤毛中具有直向同源物; 因此，我们的工作将为了解许多人类纤毛疾病的病因提供分子基础。