Molecular Analysis of Tmie in sensory hair cells

项目摘要 Transmembrane inner ear-expressed gene（TMIE）编码一种听觉必需的跨膜蛋白 和前庭功能。最近一项对Tmie-/-小鼠的研究提供了强有力的证据， TMIE在耳蜗毛细胞机械传导中的中心作用。作为转导复合体的一员， 小鼠TMIE可以结合原钙粘蛋白15的特定同种型和/或另一种膜蛋白、蛋白质 脂肪瘤HMGIC融合伴侣样5（LHFPL 5）。尽管有这些开创性的发现， TMIE作为转导复合体的一员发挥作用，迄今为止，它在前庭毛细胞中的作用是 未开发的本提案的目的是对斑马鱼Tmie进行全面研究， 鉴定（i）将Tmie定位于静纤毛，（ii）调节 机械转导，以及（iii）与转导的其他成员的遗传和生物化学相互作用 复杂.所提出的实验将利用我们收集的机械转导突变体， 我们已经在斑马鱼中开发了工具来研究Tmie在毛细胞中的功能。我们的初步数据 表明斑马鱼Tmie需要跨膜通道样1（Tmc 1）和Tmc 2b定位， 内耳毛细胞的静纤毛和侧线器。这一观察结果令人惊讶， 然而，在小鼠外毛细胞中使用Myc标记的TMC 2的实验结果表明，TMIE可能 在不同的细胞类型中发挥不同的作用。拟议的实验将扩大这些新的发现， 鉴定Tmie中促进与Lhfp 15 a相互作用的氨基酸基序以及Tmcs在Lhfp 15 a中的定位， 毛细胞中机械传导的位点。这项工作将提供对Tmie在以下方面的作用的机械见解： 毛细胞，并提供了更好地了解Tmie的分子决定因素，这是至关重要的脊椎动物 听觉和平衡

PROJECT SUMMARY Transmembrane inner ear-expressed gene (TMIE) encodes a transmembrane protein that essential for hearing and vestibular function in vertebrates. A recent study of Tmie-/- mice has provided strong evidence for a central role of TMIE in mechanotransduction in cochlear hair cells. As a member of the transduction complex, mouse TMIE can bind to a specific isoform of Protocaderin 15 and/or another membrane protein, protein Lipoma HMGIC Fusion Partner-Like 5 (LHFPL5). Despite these seminal findings, it is not well understood how TMIE functions as a member of the transduction complex, and to date, its role in vestibular hair cells is unexplored. The aim of the present proposal is to conduct a comprehensive study of zebrafish Tmie in terms of identifying the protein motifs that are required for (i) localization of Tmie to stereocilia, (ii) modulation of mechanotransduction, and (iii) genetic and biochemical interactions with other members of the transduction complex. The proposed experiments will take advantage of our collection of mechanotransduction mutants and tools we have developed in zebrafish to investigate the function of Tmie in hair cells. Our preliminary data indicate that zebrafish Tmie is required the localization of Transmembrane channel like 1 (Tmc1) and Tmc2b to the stereocilia of hair cells in the inner ear and lateral line organ. This observation is surprising in light of experiments with Myc tagged TMC2 in mouse outer hair cells, however, the results suggest that TMIE may play different roles in different cell types. The proposed experiments will expand upon these novel findings and identify the amino acid motifs in Tmie that promote interaction with Lhfpl5a and the localization of the Tmcs to the site of mechanotransduction in hair cells. This work will provide mechanistic insights into the role of Tmie in hair cells and provide a better understanding the molecular determinants of Tmie that are critical for vertebrate hearing and balance.