Molecular Mechanisms of Hair Bundle Development and Maintenance

项目摘要 检测耳蜗声需要毛细胞和它们对机械敏感的细胞器，称为 立体纤毛。这个实验室的长期目标是研究立体纤毛是如何生长的，以及它们的完整性是如何 维持了一生。这些都是关键的过程，通常以遗传性的形式被破坏 人类听力丧失。在这个提议中，我们研究了一种名为肌球蛋白15(MYO15A)的分子马达，它可以设置 肌动蛋白细丝核心的大小，它是每个立体纤毛的结构基础。基因突变 MYO15A基因导致人类遗传性耳聋，DFNB3。我们的初步实验揭示了一种新的 允许MYO15A控制肌动蛋白核心的机制，我们假设毛细胞调节 使用不同MYO15A亚型的立体纤毛结构。为了测试这一点，我们将研究分子特性 以了解它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，并研究突变如何在小鼠模型中导致听力损失。在目标1中，我们使用纯化的 广泛研究MYO15A如何加速肌动蛋白的蛋白质和光谱/单分子分析 聚合反应。作为这项工作的一部分，我们将引入突变来探索MYO15A中的候选区域 这是这项活动的基础。在目标2中，我们将我们的研究扩展到MYO15A的不同亚型，并使用生化分析 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 受监管的。在目标3中，我们描述了一只突变小鼠的特征，其中一种新的MYO15A亚型已经被使用 CRISPR基因工程，并研究这些动物如何失去他们的听力使用组合的高- 分辨电子显微镜和光学显微镜。总体而言，我们的提案将向Basic提供关键的新信息 除了揭示导致耳聋的不同病理因素外，立体纤毛可塑性的机制 患有DFNB3的患者。

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.