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.

项目摘要/摘要 听觉感觉毛细胞使用一束称为肌动蛋白的细胞蛋白转导声音 由尖端链路，顶部连接器和侧连接器以及流体力耦合在一起。活动的活动 机械电流翻译（MET）通道位于较短立体的顶部附近，已调制 通过尖端链接连接传达的立体胶质的差分运动。那就是立体膜运动调节 MET通道的开放概率将声音传播到大脑。基本目标 该提议的特征是表征立体连接的机械基础，这些连接塑造了 施加到MET通道的力。许多人类死亡基因会影响MET的分子成分 机械，包括小费链接和MET频道。组件的生物物理特征耦合 束指定它们如何过滤刺激。了解哺乳动物头发耦合的机械特性 束对于我们对毛细胞功能和听力的理解至关重要（AIM 1，2）。我们假设这一点 通道打开的概率反映了尖端链接中的张力，并且发束刚度的变化 通道门控在哺乳动物的耳蜗头发束中。为了检验这些假设，头发束 将使用新开发的技术研究机械师，该技术使用直径约1 µm的硬探针推 在1-3的立体膜上，将通过连接将其剩余的立体膜移位。高的- 速度运动跟踪将用于揭示单个立体胶质在行1的快速（<100μs）运动。 和2，允许对立体助力连通性进行表征，而全细胞电压夹提供了MET 当前响应。 MET机械及其按钙调节将通过升高或降低开放来检查 通过改变细胞内的无钙水平，破坏尖端链路连接（AIM 1），并使用 频道封锁（AIM 2）。该提案的实验，他们的分析和传播 调查结果将为申请人提供强大的技术培训，提供成为一种必要的工具 国际竞争，严格和独立的研究科学家。专业发展将是 由实验室环境中的经验，部门以及环境以及 斯坦福大学提供的资源。技术和职业发展是通过出色的 与斯坦福大学内外的杰出研究人员的讲习班，半手，会议和合作。 该提案中概述的研究培训计划旨在创造通往独立的途径 技术专长和基础数据将为独立工作提供基石。