Identification and functional analysis of A-tubule MIPs

项目总结 青少年肌阵挛癫痫(JME)是最常见的遗传性早发性癫痫，然而，其 分子病理学对此知之甚少。儿童JME最常见的突变基因编码 运动纤毛结构蛋白Rib72。这一发现提供了癫痫和癫痫之间的第一个基因联系 活动的纤毛。纤毛运动功能的一个关键功能是它们击打和移动细胞外液的能力。此外 对JME来说，睫毛功能缺陷会导致各种破坏性的人类疾病，统称为 纤毛病。这些疾病包括脑积水、呼吸窘迫以及男性和女性不孕不育。 如何构建和维持可移动的纤毛以支持持续的纤毛跳动仍然是一个关键问题 田野里的问题。活动纤毛的核心--纤毛轴丝--由两个个体组成 微管由9组二重微管环绕。作为无情地弯曲的必要条件 它们经得起考验，与细胞质对应的微管相比，双线微管具有独特的稳定性。细腻 使用单粒子冷冻电子显微镜和冷冻电子断层扫描技术进行的结构研究 纤毛轴丝的亚纳米分辨率，揭示了一组新的微管结合蛋白 位于二重微管的管腔内。这些内部密度被称为微管。 内部蛋白(MIP)，以及它们的身份和功能在很大程度上仍不清楚。在我的博士后期间 在加州大学戴维斯分校马克·威尼博士的实验室接受培训时，我发现JME相关蛋白Rib72 是将许多尚未确定的MIP募集到双线微管的A管所必需的 嗜热四膜虫纤毛轴丝。Rib72的丢失会导致活动的纤毛跳动缺陷和 轴丝稳定性受损。这些结果表明，A-微管分子印迹蛋白在血管紧张素转换酶中起着不可或缺的作用。 功能性活动纤毛的建立。为了确定依赖Rib72的A-微管MIP的身份， 我们进行了质谱学筛查，比较了野生型和RIB72基因敲除的纤毛膜四膜虫 并鉴定了一组候选的A-微管MIP。我在圣克拉拉大学的实验室与 与马克·威尼博士和大卫·阿加德博士(加州大学旧金山分校)的实验室合作，为 这些候选MIP的识别和表征。到目前为止，我们已经成功本地化并 我们已经开始研究第二个A-微管MIP，Rib43。这个 该项目的长期目标是确定A-Tubule MIP组件，了解Atubule MIP的功能作用 运动纤毛A小管MIPs及其在轴突双重体中功能的研究 微管。为了实现这些目标，我们将：1)从我们的列表中识别和本地化A管MIP 2)对这些A-微管分子印迹蛋白进行功能鉴定，包括Rib43。除了回答 关于运动纤毛结构和功能的关键问题，我们的研究可能会提供新的治疗见解 纤毛疾病，包括JME。

PROJECT SUMMARY Juvenile myoclonic epilepsy (JME) is the most common form of inherited early-onset epilepsy, however, its molecular pathology is poorly understood. The most commonly mutated gene in children with JME encodes the motile cilia structural protein, Rib72. This discovery provided the first genetic link between epilepsy and motile cilia. A key function of motile cilia function is their ability to beat and move extracellular fluid. In addition to JME, defects in ciliary function result in diverse and devastating human disorders collectively known as ciliopathies. These disorders include hydrocephaly, respiratory distress, and both male and female infertility. How motile cilia are built and maintained to support persistent ciliary beating remains a key unanswered question in the field. The core of motile cilia — called the ciliary axoneme — is composed of two individual microtubules encircled by nine sets of doublet microtubules. As a requisite for the relentless bending that they endure, the doublet microtubules are uniquely stable relative to their cytoplasmic counterparts. Exquisite structural studies using single-particle cryo-electron microscopy and cryo-electron tomography have detailed the ciliary axoneme at sub-nanometer resolution and revealed that a novel set of microtubule binding proteins resides within the lumen of the doublet microtubules. These internal densities have been termed Microtubule Inner Proteins (MIPs), and their identities and functions remain largely unknown. During my postdoctoral training in the laboratory of Dr. Mark Winey (UC Davis), I discovered that the JME-associated protein Rib72 is required for the recruitment of many of the as-yet unidentified MIPs to the A-tubule of doublet microtubules in Tetrahymena thermophila ciliary axonemes. Loss of Rib72 results in motile cilia beating defects and impaired axoneme stability. These results suggest that A-tubule MIPs play an integral role in the establishment of functional motile cilia. To determine the identities of the Rib72-dependent A-tubule MIPs, we carried out a mass spectrometry screen comparing wild-type versus RIB72 knockout Tetrahymena ciliary axonemes and identified a set of candidate A-tubule MIPs. My lab at Santa Clara University has collaborated with the labs of Dr. Mark Winey and Dr. David Agard (UCSF) to establish a research pipeline for the identification and characterization of these candidate MIPs. To date, we have successfully localized and characterized the A-tubule MIP Fap115, and we have initiated studies of a second A-tubule MIP, Rib43. The long-term goals of this project are to identify A-tubule MIP components, to understand the functional roles of A-tubule MIPs in motile cilia and to determine the functions of A-tubule MIPs within axonemal doublet microtubules. To achieve these goals, we will: 1) identify and localize the A-tubule MIPs from our list of candidates, and 2) functionally characterize these A-tubule MIPs, including Rib43. In addition to answering key questions about motile cilia structure and function, our studies may provide novel therapeutic insights into ciliopathies, including JME.