EFHC gene function in ciliary axomenes.

项目摘要： 虽然青少年肌阵挛性癫痫（JME）是遗传性青少年癫痫的最常见形式，但其 对潜在的病理学仍然知之甚少。编码运动纤毛结构的两个基因的突变 蛋白质-EFHC 1和EFHC 2-已被证明是导致JME的原因，提供了第一个基因之间的联系， 纤毛运动和癫痫运动纤毛是基于微管的细胞附属物，反复波动， 移动细胞外液。在癫痫之外，不能产生细胞外液流动导致各种各样的癫痫。 严重的人类疾病，包括原发性睫状体运动障碍、脑积水和听力损失。的 维持运动纤毛结构是纤毛功能的组成部分，纤毛生物学领域强烈关注 理解这种复杂的相互作用。例如，活动纤毛必须能够弯曲以传播节拍 然而，它们也必须足够稳定，以承受自身跳动产生的力量。运动纤毛 跳动依赖于构成它们的微管。运动纤毛轴丝由九组 修饰的双峰微管围绕中心的一对微管放射状排列。低温电子 断层扫描显示，在已被发现的双微管的管腔内的保守密度 称为微管内蛋白（MIP）。这些密度是纤毛轴丝所特有的，它们的蛋白质 组件和功能目前尚不清楚。MIPs的损失可能会影响结构 运动纤毛轴丝的完整性。关于JME连接的运动纤毛蛋白EFHC 1和EFHC 2， EFHC 2，它们是纤毛轴丝的微管相关成分。我们已经开始调查 EFHC 1和EFHC 2在水生纤毛虫Tetrahymena thermophila（Tetrahymena）中的功能。我们 发现EFHC 1和EFHC 2的四膜虫直系同源物-Bbc 73和Bbc 60分别是 轴丝蛋白质是运动纤毛跳动功能所必需的。我们也很高兴地发现，Bbc 73和 Bbc 60是形成位于轴丝偶联体A-小管中的许多MIP所必需的 微管我们进行了质谱筛选，以鉴定需要EFHC蛋白的蛋白质， 它们在四膜虫中的纤毛轴丝的定位。我们鉴定了一些感兴趣的蛋白质，包括 CAPS是一种小的钙结合蛋白，在Bbc 73和Bbc 60依赖性的细胞内定位于纤毛轴丝。 方式本项目的长期目标是：了解EFHC蛋白在运动纤毛功能中的作用; 以确定MIP组件;并确定轴丝双微管内的MIP的功能。到 为了实现这些目标，我们将：1）确定EFHC蛋白在运动纤毛中的功能和定位; 2） 鉴定需要EFHC蛋白用于其定位的运动纤毛轴丝的组分，或 接近EFHC蛋白;和3）功能表征CAPS和其他新鉴定的轴丝 是潜在的MIP成分的蛋白质。我们提出的实验结果将回答基本的 关于纤毛生物学中EFHC蛋白质的问题，并可能提供新的见解癫痫的病理学。

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.