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.

项目摘要/摘要 突变 肌球蛋白VIIA（myo7a） 是Usher综合征1型的最常见原因。 相信myo7a 对于植发器机械转导（MET）尖端链路复合物的形成和功能至关重要 立体脚踝链接，但其在这些复合物中的精度函数尚不清楚。 在初步研究中，我们发现耳蜗表达了MyO7A的多种同工型。一般 经典的同工型（myo7a-c）被专门删除的工程小鼠模型（MyO7A-ΔC小鼠）， 内毛细胞（IHC）中的MyO7a表达严重降低，在顶部外发的程度较小 细胞（OHC）。相比 OHC中的MyO7a水平的降低，沿构图轴的强度变化。 这些模型LED的分析 假设两个主要同工型在耳蜗中以完整的方式表达：IHCS 主要表达规范的同工型MyO7A-C，而替代同工型的水平要低得多 myo7a-n。在OHC中，这两种同工型以沿构件轴的相对梯度表示。 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 在以下三个目的中询问MyO7a在毛细胞中的功能作用： 在特定目标（SA）1中，我们建议进一步研究MyO7a同工型的种类及其表达 和人工耳蜗细胞中的定位。为此，我们已经生成了一个鼠标模型 同工型在遗传上标记，使我们能够确定其细胞和亚细胞定位以及 表征同工型特异性相互作用组。 在SA2中，我们将测试每种MyO7A同工型的功能意义，以满足和听力 表现。初步电生理研究表明，规范同工型的遗传缺失 影响IHC中流体射流刺激的静止概率和电流激活，一致 肌Myo7a在张紧尖端链路复合物中的推定作用。 最后，我们问为什么不同类型的毛细胞表达了MyO7a的不同同工型。为了解决这个问题，在SA3中 我们检验了myo7a同工型的差分表达来调整头发中的张力张力的假设 细胞，从而调节沿构图轴的电流特性。 该项目通过多学科的合作，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具有提供关键的潜力 深入了解重要的耳聋基因的功能。