Myosin 7a isoforms in functional diversity of cochlear hair cells

耳蜗毛细胞功能多样性中的肌球蛋白 7a 亚型

基本信息

批准号：
9925302
负责人：
Jung-Bum Shin
金额：
$ 16.15万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9925302
关键词：
Address Adopted Affect Age Amino Acids Auditory Automobile Driving Binding Sites Biochemical Cell physiology Cells Characteristics Cochlea Complex DNA Defect Development Embryonic Development Genes Genetically Engineered Mouse Hair Hair Cells Hearing Human Impairment Inner Hair Cells Introns Investigation Knock-out Knockout Mice Lead Link Location MYO7A gene Maintenance Measures Mechanics Mediating Modeling Molecular Molecular Motors Morphology Motor Activity Mus Mutation Myosin ATPase N-terminal Nucleic Acid Regulatory Sequences Organ Outcome Outer Hair Cells Output Phenotype Physiological Probability Property Protein Isoforms Recovery Regulatory Element Reporting Research Rest Role Sensory Shapes Site Specific qualifier value Testing Time Transcription Initiation Site Usher Syndrome cell type cellular engineering deaf deafness density differential expression knock-down mechanotransduction mouse model novel otoacoustic emission postnatal receptor repaired tool transcription factor

项目摘要

Abstract In the mammalian cochlea, inner hair cells (IHCs) operate as auditory receptors, while outer hair cells (OHCs) provide the cellular basis for cochlear amplification. It is well established that hair cell mechanotransduction (MET) properties, such as resting open probability, differ significantly between the two cochlear hair cell types. The molecular basis of these differences, and their significance for the respective functions of OHCs and IHCs, are not known. In preliminary studies, we discovered that two isoforms of Myosin VIIa (MYO7A) are expressed in the cochlea, generated by alternative start sites. Analysis of isoform-specific KO mice demonstrated that the longer MYO7A isoform is preferentially expressed in IHCs, while the shorter isoform, which lacks an 11-amino acid N-terminal extension, is predominantly expressed in OHCs. In mice lacking the long isoform, IHCs develop normally but progressively lose the transducing rows of stereocilia (rows 2 and 3). Additionally, these mice suffer profound hearing loss by 9 weeks of age. Remarkably, distortion product otoacoustic emissions (DPOAEs), a measure of OHC function, are unaffected, consistent with an IHC-specific phenotype. The molecular, biochemical and mouse tools we generated provide a strong platform for addressing fundamental questions regarding the function of MYO7A in hair cell MET, particularly in specifying the characteristic differences in MET properties in the two cochlear hair cell types. In Specific Aim 1, we test whether MYO7A is required for hair cell MET. Most models designate MYO7A as a molecular motor integral to the upper tip link density, possibly involved in establishing the mechanical tension important for MET function. However, direct evidence is lacking because knockout and knockdown mouse models of MYO7A have severe defects in hair bundle development. Our long isoform-specific KO mice allow the investigation of MYO7A function in normally developed hair cells. In Specific Aim 2, we will define the enzymatic activities and subcellular localization of MYO7A isoforms in IHCs and OHCs, and then correlate these outcomes with cell type-specific physiological and functional characteristics. Finally, in SA3, we propose to identify cis-and trans-regulatory factors that mediate the cell-type specific expression of MYO7A isoforms. In summary, a novel feature of MYO7A, a well- characterized deafness gene, will enable our exploration of the molecular differences between inner and outer hair cells, an issue of considerable significance for hearing research.

抽象的在哺乳动物耳蜗中，内毛细胞 (IHC) 充当听觉感受器，而外毛细胞则充当听觉感受器（OHC）为耳蜗放大提供细胞基础。众所周知，毛细胞机械传导 (MET) 特性（例如静息开放概率）之间存在显着差异两种耳蜗毛细胞类型。这些差异的分子基础及其意义 OHC 和 IHC 各自的功能尚不清楚。在初步研究中，我们发现肌球蛋白 VIIa (MYO7A) 的两种亚型在耳蜗，由替代起始位点生成。对同种型特异性 KO 小鼠的分析表明较长的 MYO7A 异构体优先在 IHC 中表达，而较短的异构体则缺乏 11 个氨基酸的 N 端延伸，主要在 OHC 中表达。在缺乏长亚型，IHC 发育正常，但逐渐失去静纤毛的转导行（第 2 行和 3）。此外，这些小鼠在 9 周龄时听力严重丧失。值得注意的是，失真产品耳声发射 (DPOAE)（OHC 功能的衡量标准）不受影响，保持一致具有 IHC 特异性表型。我们生成的分子、生化和小鼠工具为解决问题提供了强大的平台关于 MYO7A 在毛细胞 MET 中的功能的基本问题，特别是在指定两种耳蜗毛细胞类型的 MET 特性存在特征性差异。在具体目标 1 中，我们测试毛细胞 MET 是否需要 MYO7A。大多数模型将 MYO7A 指定为分子电机与上尖端链接密度不可分割，可能参与建立机械张力对于 MET 功能很重要。然而，由于敲除和击倒，缺乏直接证据 MYO7A 小鼠模型在发束发育方面存在严重缺陷。我们的长异构体特异性 KO 小鼠可以研究正常发育的毛细胞中的 MYO7A 功能。在具体目标 2 中，我们将定义 IHC 中 MYO7A 亚型的酶活性和亚细胞定位 OHC，然后将这些结果与细胞类型特异性的生理和功能相关联特征。最后，在 SA3 中，我们建议识别介导的顺式和反式调节因子 MYO7A 亚型的细胞类型特异性表达。总之，MYO7A 的一个新颖特征是，表征的耳聋基因，将使我们能够探索内在的分子差异和外毛细胞，这对于听力研究具有重要意义。