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

项目摘要/摘要 听觉感觉毛细胞利用一束充满肌动蛋白的细胞突起传递声音，这种突起被称为立体纤毛 它们通过顶端连接件、顶部连接件和侧面连接件以及流体力连接在一起。的活动 位于较短的立体纤毛顶部附近的机电传导(MET)通道被调制 通过通过尖端连杆连接传送的立体纤毛的差异运动。因此，立体纤毛运动调节 将声音传递到大脑的MET通道的开放概率。根本目标 这一建议的目的是表征立体纤毛连接的机械基础，这些连接形成了 应用于MET频道的力。许多人类耳聋基因会影响MET的分子组成 机械，包括尖端链接和MET频道。偶联的组件的生物物理特性 束决定了他们如何过滤刺激。了解哺乳动物毛发中联接器的机械特性 束对于我们理解毛细胞的功能和听力是必不可少的(目标1，2)。我们假设 通道开放概率反映了尖端链接中的张力，以及与以下相关的发束硬度的变化 通道门控将出现在哺乳动物的耳蜗毛束中。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微米的刚性探头来推动 在1-3个体纤毛上，它将通过连接它们的连接来取代剩余的体毛纤毛。高- 将使用速度运动跟踪来显示第一行中单个立体纤毛的快速(&lt；100μS)运动 以及2，允许在全电池电压钳提供MET的同时表征立体纤毛连接性 当前响应。用升降开口法检查MET机械及其钙的调节 通过改变细胞内游离钙水平，中断末端链接连接(目标1)，以及 封锁航道(目标2)。该提案中的实验、它们的分析以及它们的传播 调查结果将为申请者提供强大的技术培训，提供成为 具有国际竞争力、严谨、独立的研究科学家。职业发展将是 由实验室环境中的经验提供，该部门以及环境和 由斯坦福大学提供的资源。技术和职业发展通过卓越的 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往独立的途径，其中 技术专长和基础数据将为独立工作提供基石。

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.