Significance of Myo7a isoforms in hair cell function

Myo7a 亚型在毛细胞功能中的意义

• 批准号: 10032862
• 负责人: Jung-Bum Shin
• 金额: $ 48.98万
• 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/10032862
• 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.

项目概要/摘要 突变在 肌球蛋白 VIIa (MYO7A) 是导致 1 型 Usher 综合征的最常见原因。 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至关重要， 静纤毛踝关节连接，但其在这些复合体中的精确功能尚不清楚。 在初步研究中，我们发现耳蜗表达多种 MYO7A 亚型。 工程小鼠模型，其中典型亚型 (Myo7a-C) 被专门删除（Myo7a-ΔC 小鼠）， 内毛细胞 (IHC) 中的 MYO7A 表达严重减少，顶外毛中的表达程度较小 相比之下，替代亚型 Myo7a-N（Myo7a-ΔN 小鼠）的缺失导致了显着的结果。 OHC 中 MYO7A 水平的降低，沿音调轴的强度有所不同。 对这些模型的分析导致 假设两种主要亚型在耳蜗中以互补方式表达：IHC 主要表达典型亚型 MYO7A-C，以及较低水平的替代亚型 MYO7A-N 在 OHC 中，两种亚型沿音调轴以相反的梯度表达。 这 MYO7A 表达的令人惊讶的特征产生了一个新颖的概念框架和实验工具 通过以下三个目标探究 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 为此，我们已经生成了一个小鼠模型，其中其中之一。 同种型具有基因标记，使我们能够确定其细胞和亚细胞定位并 表征异构体特异性相互作用组。 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 初步的电生理学研究表明，典型亚型的基因缺失。 影响 IHC 中流体喷射刺激的静息开放概率和电流激活，一致 MYO7A 在拉紧尖端连接复合体方面具有假定的作用。 最后，我们询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， 我们测试了这样的假设：MYO7A 亚型的差异表达有助于调节头发的尖端连接张力 细胞，从而沿着音调轴调节 MET 电流特性。 该项目通过多学科合作实现了分子操作的结合 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 深入了解重要的耳聋基因的功能。