FORCE SPECTROSCOPY AND STRUCTURAL BIOLOGY OF HAIR-CELL TIP LINKS

描述（由申请人提供）：毛细胞尖端连接是内耳中机械传导装置的重要组成部分。在过去的四年里，我的研究主要集中在毛细胞尖端连接的结构生物学上。我计划继续这条研究路线，因为我的长期职业目标是领导一个多学科研究小组，研究毛细胞机械传导的分子机制。 我在博士后培训期间所做的研究工作回答了一些关于tip-link片段的弹性以及cadherin-23和protocadheirn-15的tips如何相互作用的关键问题 彼此之间然而，这项工作也打开了探索整个尖端链接的生物物理学的大门，并产生了关于尖端链接的强度和弹性的计算预测，这些预测需要通过实验来验证。我的短期目标是确定整个尖端链接的结构，通过实验测量尖端链接键的强度，并确定机械力如何通过尖端链接传递到毛细胞转导通道。 为了实现我的短期目标，在K99指导阶段，我将学习如何使用巴斯德毕赤酵母系统表达涉及多个EC重复序列的长细胞外末端连接片段。Gaudet实验室拥有在该系统中大规模表达蛋白质的专业知识和工具 与此同时，我将学习如何使用科里实验室的光镊装置。这包括学习如何使用和对准激光，如何设计和合成分子和功能化的微米级聚苯乙烯珠之间的连接体，以及进行实际实验。 将在两个实验室接受培训，一组杰出和特别友好的研究人员。我将直接受益于与真核蛋白质表达（高特实验室）和光学镊子（科里实验室）的使用专家的互动。我还将听取我的顾问委员会成员Wesley Wong博士的建议，他是单分子力光谱学的专家。这些实验室的所有成员将为我的培训提供完美而独特的跨学科环境。 此外，我将有机会使用科里和高德特实验室的先进设施。我将使用最先进的设备进行单分子力谱实验，真核蛋白质生产和X射线晶体学。 完成指导阶段后，我将在广泛的计算和实验技术方面拥有足够的专业知识，开始独立的职业生涯，研究整个尖端链接功能的分子机制，如下所述。 (Aim 1）光镊将用于探测由钙粘蛋白-23和原钙粘蛋白-15的尖端形成的二聚体键的强度，以及由这些分子的较长结构域形成的预期四聚体键的强度。这些实验不仅可以揭示解束缚力，而且还可以揭示中间态，并且沿着模拟，揭示与键的形成和断裂相关的分子机制。尖端连接键的断裂力将提供关于大声刺激对毛细胞功能的后果的线索。 (Aim 2）将获得整个细胞外尖端连接的结构模型，以表征异源EC重复序列，建立它们的弹性，并确定涉及多个EC重复序列的指状交错是否可行。研究结果将最终确定是否尖端链接可以毛细胞门控弹簧，并提供一个结构框架来解释多个耳聋突变。 (Aim 3）将模拟包埋在脂质双层中的原钙粘蛋白-15的跨膜结构域的机械响应，沿着具有膜门控通道，以测试力是直接还是间接通过脂质传递到毛细胞转导通道。结果将提供在计算机和体外平台，以测试模型的MS通道激活，并澄清有争议的作用，膜张力在内耳力学转导。

DESCRIPTION (provided by applicant): The hair-cell tip link is an essential component of the mechanotransduction apparatus in the inner ear. During the past four years my research has focused on the structural biology of hair-cell tip links. I plan to continue this line of research,as my long-term career goal is to lead a multidisciplinary research group that studies the molecular mechanisms of hair-cell mechanotransduction. The research work I did during my postdoctoral training answered some key questions about the elasticity of tip-link fragments, and how the tips of cadherin-23 and protocadheirn-15 interact with each other. However, this work also opened the door to explore the biophysics of the entire tip link, and produced computational predictions about the strength and elasticity of tip links tha need to be verified experimentally. My short-term goals are to determine the structure of the whole tip link, to measure the strength of the tip-link bond experimentally, and to establish how mechanical force is transmitted through the tip link and to the hair-cell transduction channel. To achieve my short term goals, and during the K99 mentored phase, I will learn how to use the Pichia pastoris system for expression of long extracellular tip link fragments involving multiple EC repeats. The Gaudet lab has the expertise and tools for large-scale expression of proteins in this system In parallel I will learn how to use the optical tweezers setup at the Corey lab. This involves learning how to use and align lasers, how to design and synthesize linkers between molecules and functionalized, micrometer-size polystyrene beads, as well as performing the actual experiments. Training will be received in two laboratories with an outstanding and exceptionally friendly group of researchers. I will directly benefit from interactions with experts in eukaryotic protein expression (Gaudet laboratory) and in the use of optical tweezers (Corey laboratory). I will also receive advice from Dr. Wesley Wong, member of my advisory committee and an expert in single-molecule force spectroscopy. All members of these laboratories will provide the perfect and unique interdisciplinary environment required for my training. In addition, I will have access to outstanding facilities at both the Corey and Gaudet laboratories. I will be using state-of-the-art equipment required for single-molecule force spectroscopy experiments, eukaryotic protein production, and X-ray crystallography. After completing the mentored phase, I will have enough expertise in a broad repertoire of computational and experimental techniques to start an independent career investigating the molecular mechanisms underlying the function of the entire tip link as outlined below. (Aim 1) Optical tweezers will be use to probe the strength of a dimeric bond formed by the tips of cadherin-23 and protocadherin-15, and of an expected tetrameric bond formed by longer domains of these molecules. The experiments may not only reveal unbinding forces, but also intermediate states and, along with simulations, molecular mechanisms associated with formation and rupture of the bond. The rupture force of the tip link bond will provide clues about the consequences of loud sound stimuli on hair cell function. (Aim 2) A structural model for the entire extracellular tip link will be obtained, to characterize heteregeous EC repeats, establish their elasticity, and determine whether interdigitation involving multiples EC repeats is feasible. Results will conclusively determine whether the tip link can be the hair-cell gating spring and provide a structural framework to interpret multiple deafness mutations. (Aim 3) The mechanical response of protocadherin-15's transmembrane domain embedded in a lipid bilayer, along with a membrane-gated channel, will be simulated to test whether force is conveyed directly, or indirectly through the lipids, to hair-cell transduction channels. Results wil provide in silico and in vitro platforms to test models of MS channel activation and clarify the controversial role of membrane tension in inner-ear mechanotransduction.