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

纤毛(也称为鞭毛)是从大多数真核细胞表面伸出的毛状细胞器，是 负责细胞运动、液体流动和感官知觉。一大组人类疾病，被称为 纤毛病变，是由纤毛功能障碍引起的。纤毛的细长形状由高度的 被称为轴丝的保守结构。在大多数活动的纤毛中，轴丝具有9+2的结构，其中 9个双线微管(DMT)围绕着中心的一对单线微管(MT)。周期性绑定 沿着每个DMT的长度是各种MT相关的蛋白质和复合体，它们装饰着 外表面和腔表面具有不同的周期(8，16，24，48和96 nm)。这些蛋白质复合体 在沿DMT的整个长度的相干寄存器中被发现，并且相干的丢失导致损害 能动性。周期性是如何建立、保持和同步的，特别是在很长的距离上 这是该领域长期存在的一个问题。每个DMT都有一个独特的结构，有一个完整的A-环- 小管和1个不完整的B小管环。DMT的独特结构是如何在体内形成的仍是个未知数 不清楚。此外，许多轴突组件在两个纵轴方向上分布不对称。 9个DMT之间的方向和径向。例如，9个DMT中有3个包含唯一的“喙” 近端B小管腔的结构。到目前为止，该基因的分子组成和生物学功能 喙是未知的。在本方案中，基于对33个微管内部蛋白(MIP)在我们的 最近的工作使用高分辨率冷冻电子显微镜(CRYO-EM)，我们建议阐明功能 衣藻突变体纤毛发生过程中的单个分子印迹点数。此应用程序的目标是使用 结合遗传和结构方法来研究管理 DMT和轴丝的组装。我们将重点关注体系结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注册的关键蛋白质 在不同的周期之间，并利用衣藻突变体研究它们之间的相互依赖 缺乏丝状分子印迹蛋白、外部螺旋卷曲蛋白和位于不同物种之间界面的蛋白。 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A管内唯一位置MT缝隙的蛋白质对B管是必不可少的 形成；(Ii)位于外连接(OJ)的分子印迹蛋白(MIP)通过屏蔽促进B管的形成 微管蛋白C末端尾部对OJ的抑制作用。(3)：我们将鉴定喙的蛋白质成分 使用高分辨率的低温电磁波。我们的初步数据显示有两种主要成分是tektin细丝 和Saxo蛋白质。我们将研究它们的细胞功能及其与人类疾病的相关性 衣藻突变株及体外检测。这里研究的大多数蛋白质在人类纤毛中都有同源基因； 因此，我们的工作将为理解许多人类纤毛疾病的病因提供分子基础。

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.