Molecular Mechanisms of Hair Bundle Development and Maintenance

发束发育和维护的分子机制

• 批准号: 10434026
• 负责人: Jonathan Edward Bird
• 金额: $ 40.7万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 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/10434026
• 关键词: ATP phosphohydrolase; ATPase Domain; 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; EPS8 gene; Ear; Electron Microscopy; Filament; Fluorescence Microscopy; Foundations; GNAI3 gene; GPSM2 gene; Genes; Genetic; Genetic Engineering; Goals; Growth; Hair; Hair Cells; Hearing; Heart; Human; In Vitro; Individual; Inherited; Laboratories; Labyrinth; Life; 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; 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; single molecule; sound; synergism; trafficking

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和它们的机械敏感细胞器，称为 静纤毛该实验室的长期目标是研究静纤毛如何生长以及它们的完整性如何 维持一辈子。这些都是关键的过程，通常在遗传性疾病中被破坏。 人类听力损失在这个提议中，我们研究了一种称为肌球蛋白15（MYO 15 A）的分子马达， 作为每个静纤毛结构基础的肌动蛋白丝核心的大小。突变 MYO 15 A基因导致人类遗传性听力损失，DFNB 3。我们最初的实验揭示了一部小说 这种机制允许MYO 15 A控制肌动蛋白核心，我们假设毛细胞调节 使用不同MYO 15 A同种型的静纤毛结构。为了验证这一点，我们将研究 了解MYO 15 A如何影响肌动蛋白核心的生长，揭示这些活动是如何调节的 在毛细胞内，并研究突变如何导致小鼠模型的听力损失。在目标1中，我们使用纯化的 蛋白质和光谱学/单分子测定可广泛表征MYO 15 A如何加速肌动蛋白 聚合法作为其中的一部分，我们将引入突变来探索MYO 15 A中的候选区域， 这一活动的基础。在目标2中，我们将我们的研究扩展到MYO 15 A的不同亚型，并使用生化测定 和冷冻电子显微镜来研究它们的酶活性的关键差异，以及这些差异是如何被 监管.在目标3中，我们描述了一种突变小鼠，其中一种新的MYO 15 A同种型已经被去除， CRISPR基因工程，并研究这些动物如何失去听力使用高- 分辨电子和光学显微镜。总的来说，我们的提案将为基本的 静纤毛可塑性的机制，除了揭示不同的病理，导致耳聋， 患有DFNB 3的患者。