Identification and functional analysis of A-tubule MIPs

A小管MIP的鉴定和功能分析

• 批准号: 10292103
• 负责人: Brian Anthony Bayless
• 金额: $ 40.77万
• 依托单位: SANTA CLARA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10292103
• 关键词: Affect; Binding; Binding Proteins; Brain; Cell surface; Cells; Child; Chlamydomonas reinhardtii; Cilia; Collaborations; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Defect; Disease; Epilepsy; Equipment; Exhibits; Extracellular Fluid; Failure; Female; Female infertility; Fluorescence; Foundations; Future; Generations; Genes; Genetic; Goals; Hand; Human; Hydrocephalus; Immunoelectron Microscopy; Impairment; Individual; Infertility; Inherited; Juvenile Myoclonic Epilepsy; Knock-out; Knowledge; Laboratories; Link; Lung; Male Infertility; Manuscripts; Mass Spectrum Analysis; Microtubules; Mutate; Organ; Phylogenetic Analysis; Play; Proteins; Proteomics; Radial; Reagent; Research; Respiratory distress; Role; San Francisco; Structural Protein; Structure; Testing; Tetrahymena; Tetrahymena thermophila; Time; Universities; appendage; base; ciliopathy; cilium motility; comparative; density; early onset; insight; interest; knock-down; link protein; male; molecular pathology; nanometer resolution; novel; novel therapeutics; particle; post-doctoral training; recruit; reproductive tract; scaffold; tool

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.

项目摘要 青少年肌阵挛性癫痫（JME）是遗传性早发性癫痫最常见的形式，然而，其 分子病理学知之甚少。JME儿童中最常见的突变基因编码 运动纤毛结构蛋白Rib 72。这一发现首次提供了癫痫与 活动纤毛运动纤毛功能的一个关键功能是它们搏动和移动细胞外液的能力。此外 对于JME来说，睫状体功能的缺陷导致多种多样的和破坏性的人类疾病，统称为 纤毛病变这些疾病包括脑积水、呼吸窘迫和男女不育。 运动纤毛是如何建立和维持以支持持续的纤毛跳动仍然是一个关键的未解之谜 在现场提问。运动纤毛的核心--纤毛轴丝--由两个独立的 微管被九组双微管包围。作为一个必要的无情的弯曲， 它们持续存在，相对于它们的细胞质对应物，双重微管是唯一稳定的。精致 使用单粒子冷冻电子显微镜和冷冻电子断层扫描的结构研究已经详细说明了 纤毛轴丝在亚纳米分辨率，并揭示了一组新的微管结合蛋白 存在于双微管的内腔中。这些内部密度被称为微管 内部蛋白质（MIPs）及其身份和功能在很大程度上仍然未知。在我的博士后期间， 在Mark Winey博士（加州大学戴维斯分校）的实验室培训中，我发现JME相关蛋白Rib 72 需要募集许多尚未鉴定的MIP到双重微管的A-微管 四膜虫纤毛轴丝。Rib 72的缺失导致运动纤毛跳动缺陷， 轴丝稳定性受损。这些结果表明，A-小管MIPs发挥不可或缺的作用， 功能性运动纤毛的建立。为了确定Rib 72依赖性A-小管MIP的身份， 我们对野生型与RIB 72敲除纤毛四膜虫进行了质谱筛选比较 轴丝，并确定了一组候选人A-小管MIP。我在圣克拉拉大学的实验室 与马克·维尼博士和大卫·阿加德博士（加州大学旧金山分校）的实验室合作，建立了一个研究管道， 鉴定和表征这些候选分子印迹聚合物。到目前为止，我们已经成功地本地化， 特征的A-小管MIP Fap 115，我们已经开始了第二个A-小管MIP，Rib 43的研究。的 本项目的长期目标是鉴定A-小管MIP组分，了解其功能作用， 运动纤毛中A-微管MIPs的表达及其在轴丝偶联体中的功能 微管为了实现这些目标，我们将：1）从我们的列表中识别和定位A-小管MIPs， 候选物，和2）功能上表征这些A-微管MIP，包括Rib 43。除了回答 关于运动纤毛结构和功能的关键问题，我们的研究可能提供新的治疗见解 包括JME