Significance of Myo7a isoforms in hair cell function

Myo7a 亚型在毛细胞功能中的意义

• 批准号: 10466879
• 负责人: Jung-Bum Shin
• 金额: $ 46.85万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10466879
• 关键词: Address; Affect; Ankle; Apical; Auditory; Binding; Biochemistry; Cell physiology; Cells; Cellular Morphology; Cochlea; Complex; Development; Electrophysiology (science); Exhibits; Genes; Genetic; Genetically Engineered Mouse; Hair; Hair Cells; Hearing; Hearing Tests; Image; Inner Hair Cells; Interdisciplinary Study; Knock-in Mouse; Link; Liquid substance; MYO7A gene; Measurement; Measures; Mechanics; Modeling; Molecular; Morphology; Motor; Mus; Mutation; Myosin ATPase; Organ; Outer Hair Cells; Output; Pattern; Performance; Probability; Property; Protein Isoforms; Proteins; Reporting; Rest; Role; Sensory; Sensory Hair; Severities; Site; Techniques; Testing; Time; Transcript; Usher Syndrome Type 1; cell type; deafness; differential expression; experimental study; insight; mechanotransduction; mouse model; novel; progressive hearing loss; response; tool

Project Summary/Abstract Mutations in myosin VIIa (MYO7A) are the most common cause of Usher syndrome type 1. MYO7A is believed to be essential for the formation and function of the hair cell mechanotransduction (MET) tip link complex and the stereocilia ankle link, but its precise function in these complexes is not known. In preliminary studies, we discovered that the cochlea expresses multiple isoforms of MYO7A. In a genetically engineered mouse model in which the canonical isoform (Myo7a-C) is specifically deleted (Myo7a-ΔC mouse), MYO7A expression is severely diminished in inner hair cells (IHCs), and to a lesser degree in apical outer hair cells (OHCs). In contrast, deletion of the alternative isoform Myo7a-N (Myo7a-ΔN mouse) led to a significant reduction of MYO7A levels in OHCs, varying in intensity along the tonotopic axis. Analyses of these models led to the hypothesis that two major isoforms are expressed in a complementary manner in the cochlea: IHCs predominantly express the canonical isoform MYO7A-C, and much lower levels of the alternative isoform MYO7A-N. In OHCs, the two isoforms are expressed in opposing gradients along the tonotopic axis. This surprising feature of MYO7A expression gave rise to a novel conceptual framework and experimental tools to interrogate the functional role of MYO7A in the hair cell in the following three aims: In Specific Aim (SA) 1, we propose to further investigate the variety of MYO7A isoforms, and their expression and localization in cochlear hair cells. To this end, we have already generated a mouse model in which one of the isoforms is genetically tagged, allowing us to determine its cellular and subcellular localization and characterize the isoform-specific interactome. In SA2, we will test the functional significance of each MYO7A isoform for hair cell MET and hearing performance. Preliminary electrophysiological studies show that genetic deletion of the canonical isoform affects resting open probability and current activation in response to fluid jet stimulation in IHCs, consistent with a putative role of MYO7A in tensioning the tip link complex. Finally, we ask why different types of hair cells express distinct isoforms of MYO7A. To address this, in SA3, we test the hypothesis that the differential expression of MYO7A isoforms serves to tune tip link tension in hair cells, thereby modulating MET current properties along the tonotopic axis. This project, through a multi-disciplinary collaboration that enabled the combination of molecular manipulations in the mouse, electrophysiology, imaging and biochemistry techniques, has the potential to provide critical insights into the function of an important deafness gene.

项目摘要/摘要 基因突变 肌球蛋白VIIA(MYO7A) 是亚瑟综合征1型最常见的原因。 MYO7A被认为 对于毛细胞机械转导(MET)尖端连接复合体的形成和功能是必不可少的 立体纤毛连接踝关节，但其在这些复合体中的确切功能尚不清楚。 在初步研究中，我们发现耳蜗表达多种MYO7A亚型。在一个基因上 特定缺失规范亚型(MYO7A-ΔC)的工程化小鼠模型(MYO7A-MYO7C小鼠)， MYO7A在内毛细胞中的表达严重减弱，在顶端外毛中的表达程度较轻 细胞(毛细胞)。相反，替代亚型MYO7A-N(MYO7A-ΔN小鼠)的缺失导致了显著的 毛细胞中MYO7A水平降低，强度沿直动轴变化。 对这些模型的分析导致 对于两种主要亚型在耳蜗中以互补的方式表达的假设：IHCs 主要表达规范的异构体MYO7A-C，以及低水平的替代异构体 MYO7A-N在毛细胞中，这两种异构体在顺位轴上以相反的梯度表达。 这 MYO7A表达的惊人特征产生了一个新的概念框架和实验工具 从以下三个方面探讨MYO7A在毛细胞中的功能作用： 在特定目的(SA)1中，我们建议进一步研究MYO7A亚型的多样性及其表达 以及耳蜗毛细胞的定位。为此，我们已经生成了一个鼠标模型，其中一个 异构体是基因标记的，使我们能够确定其细胞和亚细胞定位和 描述异构体特异性相互作用组的特征。 在SA2中，我们将测试每个MYO7A亚型对毛细胞MET和听力的功能意义 性能。初步的电生理学研究表明，典型亚型的遗传缺失 影响IHC对流体喷射刺激的静息开放概率和电流激活，一致 MYO7A在拉伸末端连接复合体中可能起到作用。 最后，我们问为什么不同类型的毛细胞表达不同的MYO7A亚型。为了解决这个问题，在SA3中， 我们验证了MYO7A亚型的差异表达有助于调节头发顶端连接张力的假设 电池，从而沿音调轴调制MET电流特性。 这个项目，通过多学科的合作，使分子操纵组合成为可能 在小鼠身上，电生理学、成像和生化技术有可能提供关键的 对一种重要的耳聋基因功能的洞察。