EFHC gene function in ciliary axomenes.

项目总结： 虽然青少年肌阵挛癫痫(JME)是遗传性青少年癫痫最常见的形式，但其 其潜在的病理机制仍然知之甚少。编码运动纤毛结构的两个基因的突变 蛋白质-EFHC1和EFHC2-已被证明引起JME，提供了第一个基因联系 运动性纤毛和癫痫。活动纤毛是以微管为基础的细胞附属物，它重复地波动到 移动细胞外液。在癫痫之外，不能产生细胞外液体流动会导致各种 严重的人类疾病，包括原发纤毛运动障碍、脑积水和听力损失。这个 纤毛运动结构的维持是纤毛功能的重要组成部分，纤毛生物学领域对纤毛的研究具有重要的意义。 理解这种复杂的相互作用。例如，活动的纤毛必须能够弯曲以传播节拍。 然而，他们也必须足够稳定，以承受自己殴打产生的力量。活动纤毛 跳动依赖于组成它们的微管。活动的纤毛轴丝由9组 改性双微管围绕一对中心微管径向排列。低温电子 断层扫描显示了双重微管管腔内的保守密度 称为微管内蛋白(MIP)。这些密度是纤毛轴丝及其蛋白质所特有的 组件和功能目前尚不清楚。MIP的流失很可能会影响到结构 活动的纤毛轴丝的完整性。人们对JME连接的游动纤毛蛋白EFHC1和EFHC1知之甚少 EFHC2，是纤毛轴丝的微管相关成分。我们已经展开了调查 探讨EFHC1和EFHC2在水生纤毛虫嗜热四膜虫(Tetrahymena Thermophila)中的功能。我们 发现EFHC1和EFHC2的四膜虫直系物-分别是Bbc73和Bbc60-是 运动纤毛搏动功能所需的轴丝蛋白。我们也很兴奋地发现Bbc73和 BBC60是形成位于轴丝二联体A管内的多个MIP所必需的 微管。我们进行了质谱学筛选，以确定需要EFHC蛋白的蛋白质 它们在四膜纲纤毛轴丝中的定位。我们确定了一些感兴趣的蛋白质，包括 CAPS，一种定位于Bbc73和Bbc60依赖的纤毛轴丝的小钙结合蛋白 举止。该项目的长期目标是：了解EFHC蛋白在运动纤毛功能中的作用； 鉴定MIP组分；并确定轴突双线微管内MIP的功能。至 为了实现这些目标，我们将：1)确定EFHC蛋白在活动纤毛中的功能和定位；2) 确定需要EFHC蛋白来定位的运动性纤毛轴丝的成分，或者是在 接近EFHC蛋白；以及3)CAPS和其他新发现的轴丝的功能特征 潜在的MIP组分的蛋白质。我们提议的实验结果将从根本上回答 关于纤毛生物学中EFHC蛋白的问题，并可能为癫痫的病理提供新的见解。

Project Summary: Although Juvenile Myoclonic Epilepsy (JME) is the most common form of inherited adolescent epilepsy, its underlying pathology remains poorly understood. Mutations in two genes that encode motile cilia structural proteins — EFHC1 and EFHC2 — have been shown to cause JME, providing the first genetic link between motile cilia and epilepsy. Motile cilia are microtubule-based cellular appendages that undulate repeatedly to move extracellular fluid. Outside of epilepsy, failure to generate extracellular fluid flow results in a variety of serious human disorders, including primary ciliary dyskinesia, hydrocephalus, and hearing loss. The maintenance of motile cilia structure is integral to cilia function, and the cilia biology field has a strong focus on understanding this complex interplay. For example, motile cilia must be able to bend to propagate a beat stroke, yet, they must also be stable enough to withstand the force generated by their own beating. Motile cilia beating relies on the microtubules that comprise them. The motile cilia axoneme consists of nine sets of modified doublet microtubules arranged radially around a central pair of microtubules. Cryo-electron tomography has revealed conserved densities within the lumen of the doublet microtubules that have been termed Microtubule Inner Proteins (MIPs). These densities are unique to ciliary axonemes, and their protein components and functions are currently unknown. It is likely that the loss of MIPs will affect the structural integrity of the motile cilia axoneme. Little is known about the JME-linked motile cilia proteins EFHC1 and EFHC2, which are microtubule-associated components of the ciliary axoneme. We have initiated investigations into the functions of EFHC1 and EFHC2 in the aquatic ciliate Tetrahymena thermophila (Tetrahymena). We discovered that the Tetrahymena orthologs of EFHC1 and EFHC2 — Bbc73 and Bbc60, respectively — are axonemal proteins required for the function of motile cilia beating. We were also excited to find that Bbc73 and Bbc60 are necessary for the formation of a number of MIPs located in the A-tubule of the axonemal doublet microtubules. We performed a mass spectrometry screen to identify proteins that require the EFHC proteins for their localization to ciliary axonemes in Tetrahymena. We identified a number of proteins of interest, including CAPS, a small calcium-binding protein that localizes to ciliary axonemes in a Bbc73- and Bbc60-dependent manner. The long-term goals of this project are: to understand the role of EFHC proteins in motile cilia function; to identify MIP components; and to determine the function of MIPs within axonemal doublet microtubules. To achieve these goals, we will: 1) determine the function and localization of EFHC proteins within motile cilia; 2) identify components of motile cilia axonemes that require EFHC proteins for their localization, or that are in close proximity to EFHC proteins; and 3) functionally characterize CAPS and other newly identified axonemal proteins that are potential MIP components. The results of our proposed experiments will answer fundamental questions about EFHC proteins in cilia biology and may provide novel insights into the pathology of epilepsy.