Mechanisms of Tip Link Tensioning in Mammalian Auditory Hair Cells

哺乳动物听觉毛细胞尖端连接张力的机制

• 批准号: 10535090
• 负责人: Abigail Dragich
• 金额: $ 4.08万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-31 至 2025-07-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10535090
• 关键词: Actins; Auditory; Biochemical; CDH23 gene; Cell Survival; Cells; Cellular biology; Clinical Treatment; Cochlea; Confocal Microscopy; Data; Detection; Development; Electron Microscopy; Electrophysiology (science); Ensure; Environment; Future; Genes; Hair; Hair Cells; Hearing; Height; Human; Image; Immobilization; In Vitro; Knock-out; Laboratories; Link; Liquid substance; MYO7A gene; Maintenance; Mechanics; Methods; Microscopy; Modeling; Molecular; Motor; Mouse Strains; Mus; Mutation; Myosin ATPase; Outer Hair Cells; Patients; Phenotype; Physiological; Play; Preparation; Principal Investigator; Proteins; Research; Rest; Role; Shapes; Slide; Speed; Structure; Techniques; base; career; deaf; deafness; density; experimental study; extracellular; hearing impairment; inhibitor; mechanotransduction; mutant; myosin VI; patch clamp; protein function; response; skills; sound

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.

项目概要 哺乳动物听觉毛细胞通过精确组织的静纤毛的偏转来检测声音 楼梯状束并通过细胞外尖端链接相互连接，声音引起的偏转调节。 尖端连杆的张力并将这些力传递到位于 即使在静止状态下，较短的静纤毛行的尖端也存在一定的张力， 确保检测到最柔和的声音并产生一些静息量的 MET 电流 我们和其他小组证明了这种静息 MET 电流可以调节。 转导静纤毛的高度，从而为长期维持 此外，我最近的研究表明，MET 依赖性的收缩。 哺乳动物听毛细胞中的静纤毛增加了 MET 机器内的张力，这只能 与经典模型相反，如果肌球蛋白不能自由移动尖端连杆的上端，就会发生这种情况 电机，但以某种方式锁定到静纤毛核心（Dragich 等人，正在审查拟议的项目）。 我们将探索这种现象的潜在分子机制。 蛋白质 C 末端 3 (GIPC3) 参与将尖端链接的上端锁定到静纤毛肌动蛋白核心。 GIPC3 的一些突变与人类听力损失有关，但这种突变的确切功能 我们的合作者（Craig Vander Kooi 博士）的数据显示，哺乳动物耳蜗中的蛋白质尚不清楚。 GIPC3 与肌球蛋白 VI (MYO6) 和上尖端连接密度 (UTLD) 蛋白 cadherin-23 相互作用 (CDH23) 和潜在的肌球蛋白 VIIa (MYO7a) 我的初步数据还表明 GIPC3 缺乏。 静息 MET 电流的丧失和耳蜗外毛 MET 反应的“滑动适应” Gipc3 中携带 p.W301X 突变的小鼠细胞，重现了已知的人类耳聋。 在项目中，我们将使用 Gipc3W301X 小鼠品系以及 Gipc3 敲除品系来确定： (a) GIPC3 在调节 MET 机制内的张力和哺乳动物听觉适应中的作用 毛细胞；(b) GIPC3 在 UTLD 组装和维持中的作用；以及 (c) 恢复的潜力； Gipc3 缺陷小鼠中的野生型 MET 反应该项目不仅将确定 GIPC3 的具体作用。 在哺乳动物听觉毛细胞中，还阐明了静纤毛维持背后的机制 MET 机械的束结构和张紧使用电生理学来处理该项目， 先进的电子显微镜和细胞生物学技术将帮助我开发一套独特的科学技术 为未来成为基础听觉研究首席研究员的职业做好准备的技能。