Molecular Analysis of Tmie in sensory hair cells

项目总结 跨膜内耳表达基因(TMIE)编码一种对听力至关重要的跨膜蛋白 以及脊椎动物的前庭功能。最近对Tmie-/-小鼠的一项研究提供了强有力的证据 TMIE在耳蜗毛细胞机械转导中的中枢作用。作为转导复合体的一员， 小鼠TMIE可以结合特定形式的原钙蛋白15和/或另一种膜蛋白 脂肪瘤HMGIC融合伙伴样蛋白5(LHFPL5)。尽管有这些开创性的发现，但人们还不太清楚 Tmie是转导复合体的一员，到目前为止，它在前庭毛细胞中的作用是 未被开发的。本建议的目的是从以下方面对斑马鱼Tmie进行全面研究 确定(I)将Tmie定位到立体纤毛所需的蛋白质基序，(Ii)调节 机械转导，以及(Iii)与转导的其他成员的遗传和生化相互作用 很复杂。拟议的实验将利用我们收集的机械转导突变株和 我们在斑马鱼中开发了一些工具来研究Tmie在毛细胞中的功能。我们的初步数据 提示斑马鱼Tmie需要跨膜通道样蛋白1(Tmc1)和Tmc2b的定位 内耳和侧线器官中毛细胞的立体纤毛。这一观察结果令人惊讶，因为 然而，用Myc标记的TMC2在小鼠外毛细胞中进行的实验表明，TMIE可能 在不同的细胞类型中扮演不同的角色。拟议的实验将在这些新发现的基础上展开，并 确定TMIE中促进与Lhfpl5a相互作用的氨基酸基序以及TMCs的定位 毛细胞中机械转导的位置。这项工作将提供对Tmie在 毛细胞，并提供更好的了解Tmie的分子决定因素，这对脊椎动物来说是关键的 听力和平衡。

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.