Myosin 7a isoforms in functional diversity of cochlear hair cells

摘要 在哺乳动物的耳蜗中，内毛细胞(IHC)作为听觉感受器运作，而外毛细胞则作为听觉感受器 (OHC)为耳蜗放大提供了细胞学基础。众所周知，毛细胞 机械传导(MET)特性，如静息开放概率，在 两种类型的耳蜗毛细胞。这些差异的分子基础，以及它们对 目前尚不清楚中心和政府间医疗中心各自的职能。 在初步研究中，我们发现肌球蛋白VIIA(MYO7A)的两种亚型在 耳蜗虫，由替代的起始部位产生。对同型异构体特异性KO小鼠的分析表明 较长的MYO7A亚型优先在IHC中表达，而较短的异构体缺乏 11个氨基酸的N-末端延伸，主要在毛细胞中表达。在缺乏Long的小鼠 IHC发育正常，但逐渐失去了立体纤毛的转导排(第2排和 3)。此外，这些小鼠在9周大时会遭受严重的听力损失。值得注意的是，失真 产品耳声发射(DPOAEs)是一种衡量OHC功能的指标，不受影响，一致 具有IHC特异性表型。 我们生成的分子、生化和鼠标工具提供了一个强大的平台 关于MYO7A在毛细胞中的功能的基本问题，特别是在指定 两种类型耳蜗毛细胞MET特性的特征差异。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 马达与上端连接密度相结合，可能与建立机械张力有关 对于仪表功能很重要。然而，缺乏直接证据，因为击倒和击倒 MYO7A小鼠模型在毛束发育方面存在严重缺陷。我们长长的同型异构体 KO小鼠允许研究正常发育的毛细胞中MYO7A的功能。在具体目标2中， 我们将确定IHC中MYO7A亚型的酶活性和亚细胞定位 然后将这些结果与特定细胞类型的生理和功能相关联 特点。最后，在SA3中，我们建议确定调节顺式和跨式调节因素 MYO7A亚型的细胞类型特异性表达。总而言之，MYO7A的一个新特点是一口井-- 特征化的耳聋基因，将使我们能够探索内在的分子差异 和外毛细胞，这是一个对听力研究具有相当重要意义的问题。

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.