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.

项目总结/摘要 突变 肌球蛋白VIIa（MYO7A） 是Usher综合征1型最常见的原因。 MYO7A被认为是 对于毛细胞机械转导（MET）尖端连接复合物的形成和功能是必需的， 静纤毛踝连接，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MYO7A的多种亚型。在一个基因 特异性缺失典型同种型（Myo7a-C）的工程小鼠模型（Myo7a-Δ C小鼠）， MYO7A的表达在内毛细胞（IHC）中严重减少，在顶外毛中程度较轻 细胞（OHC）。相比之下，替代同种型Myo7a-N（Myo7a-Δ N小鼠）的缺失导致显著的细胞凋亡。 OHC中MYO7A水平的降低，强度沿着色调轴变化。 对这些模型的分析 两种主要的同种型在耳蜗中以互补的方式表达的假设： 主要表达经典亚型MYO 7A-C，而替代亚型的水平低得多 MYO7A-N。在OHC中，两种亚型沿色调轴沿着以相反的梯度表达。 这 MYO7A表达的一个令人惊讶的特征产生了一个新的概念框架和实验工具， 在以下三个目标中询问MYO7A在毛细胞中的功能作用： 在Specific Aim（SA）1中，我们建议进一步研究MYO7A亚型的多样性及其表达， 并定位于耳蜗毛细胞。为此，我们已经生成了一个小鼠模型，其中 同种型被基因标记，使我们能够确定其细胞和亚细胞定位， 表征同种型特异性相互作用组。 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 影响静息开放概率和电流激活，以响应IHC中的液体喷射刺激， MYO7A在尖端连接复合体的张紧中具有推定的作用。 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， 我们检验了MYO7A亚型的差异表达有助于调节毛发中的尖端连接张力的假设， 细胞，从而调节沿着所述张力拓扑轴的MET电流性质。 这个项目，通过多学科的合作，使分子操作的组合， 在小鼠中，电生理学、成像和生物化学技术，有可能提供关键的 对一个重要耳聋基因功能的深入了解。