Identification and functional analysis of A-tubule MIPs

A小管MIP的鉴定和功能分析

基本信息

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
项目类别：
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

项目摘要

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.

项目概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，其对 JME 儿童中最常见的突变基因的分子病理学了解甚少。运动纤毛结构蛋白 Rib72 首次揭示了癫痫与癫痫之间的遗传联系。运动纤毛的一个关键功能是其搏动和移动细胞外液的能力。 JME 认为，纤毛功能缺陷会导致多种破坏性人类疾病，统称为这些疾病包括脑积水、呼吸窘迫以及男性和女性不育症。如何构建和维持活动纤毛以支持持续的纤毛跳动仍然是一个尚未解答的关键运动纤毛的核心——称为纤毛轴丝——由两个个体组成。微管被九组双联微管包围，这是不断弯曲的必要条件。它们经久耐用，双联体微管相对于其细胞质分子具有独特的稳定性。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详细说明了以亚纳米分辨率观察纤毛轴丝，并揭示了一组新型微管结合蛋白存在于双微管的管腔内，这些内部密度被称为微管。在我博士后期间，内部蛋白质（MIP）及其身份和功能在很大程度上仍然未知。在 Mark Winey 博士（加州大学戴维斯分校）实验室的培训中，我发现 JME 相关蛋白 Rib72 将许多尚未鉴定的 MIP 招募到双联微管的 A 管中是必需的在嗜热四膜虫纤毛轴丝中，Rib72 的缺失导致运动性纤毛跳动缺陷和这些结果表明 A 管 MIP 在轴丝稳定性中发挥着不可或缺的作用。建立功能性运动纤毛以确定 Rib72 依赖性 A 管 MIP 的身份，我们进行了质谱筛选，比较了野生型与 RIB72 敲除型四膜虫纤毛轴丝并确定了一组候选 A 管 MIP。我在圣克拉拉大学的实验室进行了合作。与 Mark Winey 博士和 David Agard 博士（加州大学旧金山分校）的实验室合作，为这些候选 MIP 的识别和表征迄今为止，我们已经成功地本地化和表征。表征了 A 管 MIP Fap115，我们已经开始研究第二个 A 管 MIP，Rib43。该项目的长期目标是识别 A 管 MIP 组件，了解 A 管 MIP 的功能作用运动纤毛中的 A 管 MIP 并确定轴丝双联内 A 管 MIP 的功能为了实现这些目标，我们将：1）从我们的列表中识别并定位 A 管 MIP。候选者，2) 除了回答之外，还对这些 A 管 MIP 进行功能表征，包括 Rib43。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纤毛病，包括 JME。