EFHC gene function in ciliary axomenes.

EFHC 基因在睫状轴丝中的功能。

• 批准号: 9900028
• 负责人: MARK WINEY
• 金额: $ 31.4万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9900028
• 关键词: Address; Adolescent; Affect; Alleles; Biology; Brain; Calcium-Binding Proteins; Cell surface; Cells; Cilia; Cilium Microtubule; Collaborations; Complex; Cryo-electron tomography; Data; Defect; Disease; Environment; Epilepsy; Equipment; Exhibits; Extracellular Fluid; Eye; Failure; Family; Genes; Genetic; Goals; Hair; Hand; Human; Hydrocephalus; Inherited; Investigation; Juvenile Myoclonic Epilepsy; Kidney; Knock-out; Lead; Link; Liquid substance; Lung; Maintenance; Mass Spectrum Analysis; Microtubules; Mus; Mutation; Myoclonus; Organ; Orthologous Gene; Pathogenesis; Pathology; Pathway interactions; Play; Point Mutation; Predisposition; Primary Ciliary Dyskinesias; Proteins; Proteomics; Radial; Reagent; Role; Seizures; Speed; Stroke; Structural Protein; Structure; Suggestion; Swimming; Tertiary Protein Structure; Testing; Tetrahymena; Tetrahymena thermophila; appendage; base; cell motility; cell type; ciliopathy; cilium motility; density; design; experimental study; fluid flow; gene function; hearing impairment; insight; interest; mutant; novel; novel therapeutics; protein structure function

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.

项目摘要： 虽然少年肌阵挛性癫痫（JME）是遗传癫痫的最常见形式，但 潜在的病理学仍然知之甚少。编码纤毛结构的两个基因的突变 蛋白质 - EFHC1和EFHC2 - 已显示出引起JME，提供了第一个遗传联系 纤毛和癫痫。纤毛运动是基于微管的细胞附属物，反复膨胀至 移动细胞外液。在癫痫以外，未能产生细胞外液流量会导致多种 严重的人类疾病，包括原发性睫状运动障碍，脑积水和听力损失。 维持纤毛纤毛结构的维护是纤毛功能不可或缺的，纤毛生物学领域非常关注 了解这个复杂的相互作用。例如，Motori Cilia必须能够弯曲以传播节拍 但是，他们还必须足够稳定，以承受自己的殴打产生的力量。纤毛纤毛 打击依赖于构成它们的微管。母亲Cilia Axoneme由九组组成 修饰的双线微管从根本上排列在中央微管周围。冷冻电子 断层扫描揭示了多次微管腔内的保守密度 称为微管内蛋白（MIPS）。这些密度是睫状轴突及其蛋白质独有的 组件和功能当前未知。 MIP的损失可能会影响结构 母亲纤毛轴突的完整性。关于JME连接的母亲纤毛蛋白EFHC1和 EFHC2，是纤毛轴突的微管相关成分。我们已经开始调查 在水生纤毛四膜热嗜热（四膜）中的EFHC1和EFHC2的功能中。我们 发现EFHC1和EFHC2的四膜直系同源物分别是BBC73和BBC60 - 是 纤毛蛋糕的功能所需的轴突蛋白。我们也很高兴发现BBC73和 BBC60对于形成位于轴突双线a小管中的许多MIP的形成必需 微管。我们进行了质谱屏幕，以识别需要EFHC蛋白的蛋白质 它们定位于四氢菌中的睫状轴突。我们确定了许多感兴趣的蛋白质，包括 盖，一种小钙结合蛋白，将定位于BBC73和BBC60依赖性的纤毛轴突 方式。该项目的长期目标是：了解EFHC蛋白在纤毛功能中的作用； 识别MIP组件；并确定轴突双线微管中MIP的功能。到 实现这些目标，我们将：1）确定纤毛中EFHC蛋白的功能和定位； 2） 确定需要EFHC蛋白来定位的母纤毛轴突的成分，或 靠近EFHC蛋白； 3）功能表征帽子和其他新鉴定的轴突 潜在MIP成分的蛋白质。我们提出的实验的结果将回答基本 关于纤毛生物学中EFHC蛋白的问题，可能会提供有关癫痫病理学的新见解。