Myosin 7a isoforms in functional diversity of cochlear hair cells

摘要 在哺乳动物耳蜗中，内毛细胞（IHC）作为听觉感受器，而外毛细胞作为听觉感受器， OHC为耳蜗放大提供细胞基础。众所周知，毛细胞 机械转导（MET）特性，如静息开放概率，在 两种耳蜗毛细胞类型。这些差异的分子基础，以及它们对 OHC和IHC各自的职能尚不清楚。 在初步的研究中，我们发现肌球蛋白VIIa（MYO 7A）的两种亚型表达于心肌细胞中， 耳蜗，由替代起始位点产生。同种型特异性KO小鼠的分析表明， 较长的MYO 7A亚型优先在IHC中表达，而较短的亚型， 11个氨基酸的N-末端延伸，主要在OHC中表达。在缺乏长 在同种型中，IHC正常发育，但逐渐失去静纤毛的转导行（第2行和第3行）。 3）。此外，这些小鼠在9周龄时遭受严重的听力损失。值得注意的是， 产品耳声发射（DPOAE），OHC功能的测量，不受影响，一致 具有IHC特异性表型。 我们产生的分子、生物化学和小鼠工具为解决这些问题提供了一个强大的平台。 关于MYO 7A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中MET特性的特征性差异。在具体目标1中， 测试MYO 7A是否需要毛细胞MET。大多数模型将MYO 7A指定为分子 电机集成到上尖端链接密度，可能涉及建立机械张力 对MET功能至关重要。然而，由于击倒和击倒， MYO 7A的小鼠模型在毛束发育中具有严重缺陷。我们的长同种型特异性 KO小鼠允许研究MYO 7A在正常发育的毛细胞中的功能。在具体目标2中， 我们将确定MYO 7A亚型在IHC中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生理和功能相关， 特色最后，在SA 3中，我们提出鉴定介导细胞凋亡的顺式和反式调节因子。 MYO 7A亚型的细胞类型特异性表达。总之，MYO 7A的一个新功能，一个很好的- 特征性耳聋基因，将使我们能够探索内部 和外毛细胞，这是一个对听力研究具有重要意义的问题。

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.