Investigating the role of mechanotransduction machinery and the rootlet in modulating stereocilia motion.

研究机械传导机制和细根在调节静纤毛运动中的作用。

• 批准号: 10676417
• 负责人: Jamis McGrath
• 金额: $ 6.95万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676417
• 关键词: Actins; Affect; Auditory; Biochemical; Brain; Buffers; Calcium; Cell physiology; Cells; Cochlea; Collaborations; Communication; Coupled; Coupling; Data; Dependence; Detection; Development; Diameter; Educational workshop; Elasticity; Elements; Environment; Functional disorder; Genes; Goals; Hair; Hair Cells; Hearing; Human; Image; Individual; International; Laboratories; Left; Link; Liquid substance; Measures; Mechanics; Mediating; Methods; Modeling; Molecular; Monitor; Motion; Movement; Noise-Induced Hearing Loss; Organ; Organelles; Pathway interactions; Probability; Productivity; Property; Proteins; Proxy; Quality of life; Regulation; Research; Research Personnel; Research Training; Resolution; Resources; Role; Scientist; Sensory Hair; Shapes; Side; Signal Transduction; Site; Speed; Stimulus; Sum; Technical Expertise; Techniques; Technology; Testing; Therapeutic Intervention; Time; Training; Tubocurarine; Universities; Work; age related; biophysical properties; career; career development; cellular transduction; deafness; design; experience; experimental study; extracellular; hearing impairment; mechanical properties; mechanotransduction; meter; patch clamp; preventive intervention; response; sound; success; symposium; tool; voltage clamp

Project Summary/Abstract Auditory sensory hair cells transduce sound using a bundle of actin-filled cellular protrusions called stereocilia which are coupled together by tip links, top connectors and side connectors, and fluid forces. Activity of mechanoelectrical transduction (MET) channels, located near the tops of the shorter stereocilia, are modulated by the differential motion of stereocilia as conveyed via the tip link connection. Thus, stereocilia motion regulates the open probability of MET channels which drives communication of sound to the brain. The fundamental goal of this proposal is to characterize the mechanical underpinnings of the stereociliary connections that shape the force applied to MET channels. Many human deafness genes affect the molecular components of the MET machinery, including tip links and MET channels. The biophysical characteristics of components coupling the bundle dictate how they filter stimuli. Understanding the mechanical properties of coupling in mammalian hair bundles is essential to our understanding of hair cell function and hearing (Aim 1, 2). We hypothesize that channel open probability reflects tension in the tip link and that changes in hair bundle stiffness associated with channel gating will be present in mammalian cochlear hair bundles. To test these hypotheses, hair bundle mechanics will be investigated using newly developed technology that uses a ~1 µm diameter stiff probe to push on 1-3 stereocilia which will displace the remaining stereocilia through the connections coupling them. High- speed motion tracking will be used to reveal the rapid (<100 μs) movements of individual stereocilia in rows 1 and 2, allowing for characterization of stereociliary connectivity while whole cell voltage clamp provides the MET current response. The MET machinery and its regulation by calcium will be examined by raising or lowering open probability by changing intracellular free calcium levels, disrupting the tip link connections (Aim 1), and with channel blockade (Aim 2). The experiments in this proposal, their analyses, and the dissemination of their findings will serve as strong technical training for the applicant, providing the tools necessary to become an internationally competitive, rigorous, and independent research scientist. Professional development will be provided by experiences within the laboratory setting, the department, as well as by the environment and resources provided by Stanford University. Technical and career development are provided through excellent workshops, seminars, conferences, and collaborations with outstanding researchers inside and outside Stanford. The research training plan outlined in this proposal is designed to create a pathway to independence where both the technical expertise and foundational data will provide the cornerstone for independent work.

项目概要/摘要 听觉感觉毛细胞利用一束充满肌动蛋白的细胞突起（称为静纤毛）来传递声音 它们通过尖端连杆、顶部连接器和侧面连接器以及流体力耦合在一起。活动 位于较短静纤毛顶部附近的机电传导 (MET) 通道受到调节 通过通过尖端连杆连接传递的静纤毛的差速运动。因此，静纤毛运动调节 驱动声音与大脑通讯的 MET 通道的开放概率。根本目标 该提案的目的是描述塑造立体纤毛连接的机械基础 施加到 MET 通道的力。许多人类耳聋基因影响 MET 的分子成分 机械，包括尖端链接和 MET 通道。耦合成分的生物物理特性 捆绑决定了他们如何过滤刺激。了解哺乳动物毛发耦合的机械特性 束对于我们了解毛细胞功能和听力至关重要（目标 1、2）。我们假设 通道打开概率反映了尖端连接的张力以及与相关的发束刚度的变化 通道门控将存在于哺乳动物耳蜗毛束中。为了检验这些假设，发束 将使用新开发的技术来研究力学，该技术使用直径约 1 µm 的刚性探针来推动 位于 1-3 个静纤毛上，这将通过耦合它们的连接取代剩余的静纤毛。高的- 速度运动跟踪将用于揭示第 1 行中单个静纤毛的快速（<100 μs）运动 2，允许表征立体纤毛连接性，同时全细胞电压钳提供 MET 当前的响应。 MET 机械及其对钙的调节将通过升高或降低开放来检查 通过改变细胞内游离钙水平、破坏尖端连接（目标 1）以及 通道封锁（目标 2）。本提案中的实验、分析及其传播 研究结果将为申请人提供强有力的技术培训，为成为一名技术人员提供必要的工具。 有国际竞争力、严谨、独立的研究科学家。专业发展将 由实验室环境、部门以及环境和环境中的经验提供 斯坦福大学提供的资源。通过优秀的技术和职业发展 研讨会、研讨会、会议以及与斯坦福大学内外杰出研究人员的合作。 本提案中概述的研究培训计划旨在创建一条通向独立的途径，其中双方 技术专长和基础数据将为独立工作提供基石。