Mechanisms of Tip Link Tensioning in Mammalian Auditory Hair Cells

项目摘要 哺乳动物的听觉毛细胞通过立体纤毛的偏转来检测声音，这些偏转是以精确的方式组织的 阶梯状的维管束，并通过细胞外顶端连接相互连接。声音诱导的偏转调制了 并将这些力传递到位于以下位置的机电转换(MET)通道 较短的一排立体纤毛的尖端。即使在休息时，顶端的链节上也有一定的张力， 其确保检测到最柔和的声音，并产生一定的静止量的仪表电流 不断地进入牢房。我们和其他小组证明，这种休眠符合电流调节 换能器立体纤毛的高度，从而提供了一种合理的机制来长期维持 立体纤毛束的形状。此外，我最近的研究表明，依赖于MET的退缩 哺乳动物听觉毛细胞中的立体纤毛增加了MET机械内的张力，这只能 与经典模型不同，如果顶端链接的上端不能被肌球蛋白自由移动，就会发生这种情况 而是以某种方式锁定在立体纤毛核心上(Dragich et.Al.，正在审查中)。拟议中的项目 将探索这一现象的潜在分子机制。我们假设Gα相互作用 蛋白质C末端3(GIPC3)参与将顶端连接的上端锁定到立体纤毛肌动蛋白核心。 GIPC3的几个突变与人类听力损失有关，但这一突变的确切功能 哺乳动物耳蜗中的蛋白质目前还不清楚。我们的合作者(Craig Vander Kooi博士)的数据显示 GIPC3与肌球蛋白VI(MYO6)和上端连接密度(UTLD)蛋白Cad-23相互作用 (CDH23)和潜在的肌球蛋白VIIa(MYO7A)。我的初步数据还显示，GIPC3缺陷导致 静息MET电流的丧失和耳蜗外毛MET反应的“滑动适应” 在Gipc3中携带p.W301X突变的小鼠细胞，重述了一种已知的人类耳聋。在这 项目中，我们将使用此Gipc3W301X小鼠品系以及Gipc3基因敲除品系来确定：(A) GIPC3在哺乳动物听觉机械张力调节和适应中的作用 毛细胞；(B)GIPC3在UTLD组装和维护中的作用；以及(C)修复的可能性 野生型对Gipc3缺陷小鼠的反应。该项目不仅将确定GIPC3的具体作用 在哺乳动物的听觉毛细胞中，也阐明了维持体毛纤毛的机制 MET机械的捆扎结构和张拉。使用电生理学来研究这个项目， 先进的电子显微镜和细胞生物学技术将帮助我发展一套独特的科学 为将来成为基础听觉研究首席研究员的职业生涯做准备的技能。

Project Summary Mammalian auditory hair cells detect sound through deflections of stereocilia that are organized in precise staircase-like bundles and interconnected by extracellular tip links. Sound-induced deflections modulate the tension of tip links and convey these forces to mechano-electrical transduction (MET) channels located at the tips of the shorter rows of stereocilia. Even at rest, there is a certain amount of tension on the tip links, which ensures detection of the softest sounds and results in some resting amount of MET current continuously entering the cell. We and other groups demonstrated that this resting MET current regulates the height of transducing stereocilia, thereby providing a plausible mechanism for long-term maintenance of the shape of stereocilia bundle. Furthermore, my recent study revealed that MET-dependent retraction of stereocilia in mammalian auditory hair cells increases the tension within MET machinery, which could only occur if, in contrast to the classical models, the upper end of the tip link is not freely moved by myosin motors but instead somehow locked to the stereocilia core (Dragich et. al., in review). The proposed project will explore a potential molecular mechanism of this phenomenon. We hypothesize that Gα-Interacting Protein, C-terminus-3 (GIPC3) is involved in locking the upper end of the tip link to the stereocilia actin core. Several mutations in GIPC3 have been linked to hearing loss in humans, but the exact function of this protein in the mammalian cochlea is yet unknown. Data from our collaborator (Dr. Craig Vander Kooi) show that GIPC3 interacts with myosin VI (MYO6) and the upper tip link density (UTLD) proteins, cadherin-23 (CDH23) and potentially myosin VIIa (MYO7a). My preliminary data also show that GIPC3 deficiency results in the loss of resting MET current and “slipping adaptation” of the MET responses in cochlear outer hair cells of mice carrying the p.W301X mutation in Gipc3, recapitulating a known human deafness. In this project, we will use this Gipc3W301X mouse strain as well as a Gipc3 knockout strain to determine: (a) the role of GIPC3 in regulating the tension within the MET machinery and adaptation in mammalian auditory hair cells; (b) the role of GIPC3 in UTLD assembly and maintenance; and (c) the potential for restoring wildtype MET responses in Gipc3-deficient mice. This project will not only identify the specific role of GIPC3 in mammalian auditory hair cells but also elucidate the mechanisms behind the maintenance of stereocilia bundle structure and tensioning of the MET machinery. Approaching this project using electrophysiology, advanced electron microscopy, and cell biology techniques will help me to develop a unique set of scientific skills in preparation for a career as a future principal investigator in basic auditory research.