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.

项目总结/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）来发声 它们通过尖端连接件、顶部连接件和侧部连接件以及流体力连接在一起。活性 位于较短静纤毛顶部附近的机械电转导（MET）通道被调节 通过静纤毛的差动运动，如通过尖端连杆连接传送的。因此，静纤毛运动调节 MET通道的开放概率，它驱动声音与大脑的通信。根本目标 该建议的主要目的是表征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的分子组成部分 机械，包括尖端链接和MET通道。生物物理特性的组件耦合 bundle束dictate决定how they filter过滤stimulus刺激.了解哺乳动物毛发中耦合的机械特性 束是必不可少的，我们的毛细胞功能和听力的理解（目的1，2）。我们假设 通道开放概率反映了尖端连接中的张力， 通道门控将存在于哺乳动物耳蜗毛束中。为了验证这些假设， 力学将使用新开发的技术进行研究，该技术使用直径约1微米的刚性探针推动 在1-3个静纤毛上，这将通过连接它们的连接取代剩余的静纤毛。高- 速度运动跟踪将用于显示第1行中个体静纤毛的快速（<100 μs）运动 和2，允许表征立纤毛连接性，而全细胞电压钳提供MET 当前响应MET机制及其钙调节将通过升高或降低开放式 通过改变细胞内游离钙水平，破坏头端连接（目标1）， 通道阻断（Aim 2）。该提案中的实验、分析和传播 调查结果将作为申请人的强有力的技术培训，提供必要的工具，成为 具有国际竞争力，严谨和独立的研究科学家。专业发展将是 由实验室环境、部门以及环境中的经验提供， 资源由斯坦福大学提供。技术和职业发展是通过优秀的 工作坊，研讨会，会议，并与斯坦福大学内外的杰出研究人员合作。 本提案中概述的研究培训计划旨在创建一条通往独立的途径， 技术专长和基础数据将为独立工作提供基石。