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 少年肌阵挛性癫痫（JME）是遗传早期癫痫的最常见形式，但是 分子病理学知之甚少。 JME编码儿童中最常见的突变基因 母纤毛结构蛋白Rib72。这一发现提供了癫痫和 Mothere Cilia。 Mothere纤毛功能的关键功能是它们击败和移动细胞外流体的能力。此外 对于JME，睫状功能的缺陷导致潜水和毁灭性的人类疾病统称为 纤毛病。这些疾病包括脑畸形，呼吸窘迫以及男性和女性不育症。 苏里亚妈妈的建造方式如何支持持续的睫毛跳动仍然是未回答的关键 在现场的问题。纤毛母亲的核心（称为睫状轴突）由两个个体组成 由九组Doublet微管包围的微管。作为远程弯曲的必要条件 它们忍受了双重微管相对于其细胞质对应物的独特稳定。精美的 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详细 亚纳米分辨率下的睫状轴突蛋白，并揭示了一组新型的微管结合蛋白 位于双线微管的管腔内。这些内部密度称为微管 内部蛋白质（MIP）及其身份和功能在很大程度上未知。在我的博士后 在Mark Winey博士（UC Davis）实验室的培训，我发现与JME相关的蛋白质RIB72 招募了许多尚未确定的MIPS招募到Doublet微纤维的A-细胞 在四膜热纤维纤维轴突中。 RIB72的损失导致苏里亚母亲击败缺陷和 轴突稳定性受损。这些结果表明，a-小管MIP在 建立功能性纤毛。为了确定依赖RIB72的A-管MIP的身份， 我们进行了一个质谱屏幕，比较了野生型与RIB72敲除四方睫状 轴突并鉴定了一组候选A-管MIP。我在圣克拉拉大学的实验室合作 在Mark Winey博士和David Agard博士（UCSF）的实验室中，建立了一条研究管道 这些候选MIP的识别和表征。迄今为止，我们已经成功本地化了， 表征了A-A-Tubule MIP FAP115，我们启动了第二个A-Pubule MIP Rib43的研究。这 该项目的长期目标是识别a-pubule MIP组件，以了解 A-Tube MIPS中的纤毛中的A-Tube MIPS确定A轴突双线内A管MIP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并定位A-Tubele MIP 候选物和2）在功能上表征了这些A-管MIP，包括RIB43。除了回答 关于跑步纤毛结构和功能的关键问题，我们的研究可能会提供新颖的热洞察力 进入包括JME在内的纤毛病。