Molecular Mechanisms of Hair Bundle Development and Maintenance

发束发育和维护的分子机制

基本信息

批准号：
10643931
负责人：
Jonathan Edward Bird
金额：
$ 39.38万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10643931
关键词：
ATP phosphohydrolase ATPase Domain Acceleration Actins Affect Animals Architecture Auditory Binding Biochemical Biological Assay C-terminal Cells Clustered Regularly Interspaced Short Palindromic Repeats Cochlea Complex Critical Pathways Cryoelectron Microscopy Cytoskeleton Data Defect Detection Development Ear Electron Microscopy Epidermal Growth Factor Receptor Pathway Substrate 8 Filament Fluorescence Microscopy Foundations GNAI3 gene GPSM2 gene Genes Genetic Genetic Engineering Goals Growth Hair Hair Cells Hearing Heart Height Human In Vitro Individual Inherited Laboratories Labyrinth Life Life Cycle Stages Longevity Maintenance Measures Mechanics Microfilaments Microscopy Molecular Molecular Motors Motor Mus Mutant Strains Mice Mutation Myosin ATPase N-terminal Organelles Outcome Pathologic Pathology Pathway interactions Patients Phase Polymers Process Property Protein Isoforms Proteins Pyrenes Resolution Sensory Spectrum Analysis Stereocilium Structure Tail Testing WHRN gene Work deafness disability experimental study hearing impairment hereditary hearing loss in vitro activity in vivo light microscopy mechanotransduction monomer mouse model mutant novel particle polymerization postnatal preservation protein complex protein purification single molecule sound synergism trafficking

项目摘要

Project Summary The detection of sound in the cochlea requires hair cells and their mechano-sensitive organelles, called stereocilia. The long-term goal of this laboratory is to study how stereocilia grow and how their integrity is maintained over a lifetime. These are critical processes and are commonly disrupted in hereditary forms of human hearing loss. In this proposal, we investigate a molecular motor called myosin 15 (MYO15A) that sets the size of the actin filament core that is the structural foundation within each stereocilium. Mutations in the MYO15A gene cause human hereditary hearing loss, DFNB3. Our initial experiments have revealed a novel mechanism that allows MYO15A to control the actin core, and we hypothesize that the hair cell regulates stereocilia architecture using different MYO15A isoforms. To test this, we will investigate the molecular properties of MYO15A to understand how it influences growth of the actin core, reveal how these activities are regulated within the hair cell, and examine how mutations cause hearing loss in a mouse model. In Aim 1, we use purified proteins and spectroscopy / single-molecule assays to extensively characterize how MYO15A accelerates actin polymerization. As part of this, we will introduce mutations to explore candidate regions within MYO15A that underlie this activity. In Aim 2, we expand our study to different isoforms of MYO15A and use biochemical assays and cryo-electron microscopy to investigate key differences in their enzymatic activity and how these are regulated. In Aim 3, we characterize a mutant mouse where a novel MYO15A isoform has been removed using CRISPR genetic engineering, and study how these animals lose their hearing using a combination of high- resolution electron and light microscopy. Overall, our proposal will provide critical new information into basic mechanisms of stereocilia plasticity, in addition to revealing the distinct pathologies that cause deafness in patients suffering with DFNB3.

项目摘要耳蜗中声音的检测需要毛细胞及其机械敏感细胞器，称为立体胶质。该实验室的长期目标是研究立体尾层如何增长以及它们的完整性如何维持一生。这些是关键过程，通常以遗传形式的破坏人类听力损失。在此提案中，我们研究了一个称为肌球蛋白15（MyO15a）的分子电机，该电动机设置为肌动蛋白细丝核的大小是每个立体核中的结构基础。突变 MyO15a基因会导致人类遗传性听力损失，DFNB3。我们最初的实验揭示了一本小说允许MyO15A控制肌动蛋白核的机制，我们假设毛细胞调节使用不同的MyO15A同工型的立体胶体体系结构。为了测试这一点，我们将研究分子特性 Myo15a了解它如何影响肌动蛋白核心的增长，揭示如何调节这些活动在毛细胞内，并检查突变如何在小鼠模型中导致听力损失。在AIM 1中，我们使用纯化蛋白质和光谱 /单分子测定法，以广泛的特征肌动蛋白如何加速肌动蛋白聚合。为此，我们将引入突变，以探索Myo15a中的候选区域这是这项活动的基础。在AIM 2中，我们将研究扩展到MyO15A的不同同工型并使用生化测定和冷冻电子显微镜，以研究其酶活性的关键差异以及它们是如何的受监管。在AIM 3中，我们表征了一种突变的小鼠，其中新颖的Myo15a同工型已使用 CRISPR基因工程，并研究这些动物如何使用高级的组合来失去听力分辨率电子和光学显微镜。总体而言，我们的建议将为基本提供关键的新信息立体胶质可塑性的机制除了揭示引起耳聋的不同病理患有DFNB3的患者。