Elucidating the architecture and composition of the hair cell mechanotransduction complex

项目摘要 位于内耳毛束尖端的机械转导机械负责我们的 运动和声音的感觉。每束由约100个排列成阶梯状的立体纤毛组成 它们通过末端连接、由原钙粘蛋白15(PCDH15)组成的细胞外蛋白丝和 钙粘蛋白-23(CDH23)。声音和流体运动使发束偏转，导致发束张开 机械转导通道(MEC)复合体位于下端-连接插入部位，导致电 信号。除了尖端连接蛋白外，MEC复合体还有三个可能的成分：脂肪瘤 HMGIC融合伙伴样蛋白LHFPL5(也称为TMHS)，跨膜内耳蛋白TMIE， 以及跨膜型通道蛋白TMC1/2，它们可能是复合体的成孔亚基。 虽然已知这些蛋白质相互作用形成听力所必需的机械转导复合体， 这种复合体的确切成分、化学计量和结构仍然难以捉摸。这项提案的目标是 是利用单分子技术和冷冻技术来阐明MEC复合体的组成和结构。 电子断层摄影术。拟议工作的主要挑战是MEC综合体的丰度较低 天然组织。我们已经开发出针对四种MEC复杂成分中的三种的高亲和力抗体 这将是我们解决这一问题的办法中的关键要素。在第一个目标中，我将利用这些抗体 用单分子下拉方法检测小鼠耳蜗毛细胞中的MEC复合体 评价MEC复合体的组成。光漂白和单分子定量实验将允许 Me测定MEC复合体的化学计量学，并测量每个耳蜗中MEC复合体的数量。在 这项提议的第二个目的是，我将使用冷冻电子断层扫描来阐明MEC复合体的结构。 我已经开发了一种方法，在玻璃冰中用立体纤毛制备冷冻-EM栅格，并获得了有希望的结果 初步体层摄影术。我将使用我们的抗体与量子点相结合，将MEC复合体标记在冷冻- EM网格并确定综合体的3D结构。说明MEC的架构和组成 复合体将为毛细胞机械转导和开放的难以捉摸的机制提供有价值的见解 为聋人开发新的治疗方法和治疗策略的大门。

Project Summary Mechanotransduction machinery located at the tips of hair bundles in the inner ear are responsible for our sensations of movement and sound. Each bundle is composed of ~100 stereocilia organized in a staircase array that are connected by tip-links, extracellular protein filaments composed of protocadherin 15 (PCDH15) and cadherin-23 (CDH23). Deflection of the hair bundle by sound and fluid movement leads to opening of the mechanotransduction channel (MEC) complex located at the lower tip-link insertion site, resulting in an electrical signal. There are three putative components of the MEC complex aside from the tip-link proteins: the lipoma HMGIC fusion partner-like 5 protein LHFPL5 (also known as TMHS), the transmembrane inner ear protein TMIE, and the transmembrane-like channel proteins TMC1/2, which are the likely pore-forming subunits of the complex. Although it is known that these proteins interact to form a mechanotransduction complex necessary for hearing, the precise composition, stoichiometry, and structure of this complex remain elusive. The goal of this proposal is to elucidate the composition and architecture of the MEC complex using single-molecule techniques and cryo- electron tomography. The main challenge of the proposed work is the low abundance of the MEC complex in native tissue. We have developed high affinity antibodies against three of the four MEC complex components that will be key elements in our approach to overcome this issue. In the first aim, I will utilize these antibodies to pull-down and detect the MEC complex from mouse cochlear hair cells using a single molecule pulldown method to assess MEC complex composition. Photobleaching and single-molecule quantitation experiments will allow me to determine MEC complex stoichiometry and measure the number of MEC complexes per cochlea. In the second aim of this proposal, I will elucidate the architecture of the MEC complex using cryo-electron tomography. I have developed a method to prepare cryo-EM grids with stereocilia in vitreous ice and obtained promising preliminary tomograms. I will use our antibodies conjugated to quantum dots to label the MEC complex on cryo- EM grids and determine a 3D structure of the complex. Illuminating the architecture and composition of the MEC complex will provide valuable insight into the elusive mechanism of hair cell mechanotransduction and open doors for the development of new therapeutics and treatment strategies for deaf individuals.