Project B

我们的听觉和平衡感依赖于内耳毛细胞顶端表面的毛束的适当发育、功能和维持。发束是由不同的具有特定功能和结构的多蛋白质复合体组成的，它们共同工作，负责机电转导、适应和可能的信号放大。这些机器中的任何一个的功能失效都会导致耳聋或不同程度的听力和平衡障碍。 我们的目标是将这些立体纤毛分子机器在其天然细胞环境中以分子分辨率可视化。我们的理论基础是，确定这些立体纤毛复合体的分子三维组织最终将导致对这种精密细胞器的发育、功能和维护的机械性理解。具体地说，我们特别建议研究低等脊椎动物(青蛙、斑马鱼)和高等脊椎动物(小鼠、豚鼠)的立体纤毛的四个区域，每个区域都有一种特定类型的多蛋白复合体，并且在毛束的形成、功能和/或维持中发挥着不同的作用。这四个地区是1。)尖端复合体，2。)肌动蛋白核心，3。)适应复合体，以及4.)锥形区域和细根。 我们的具体目标如下： 具体目标1：确定不育纤毛顶端复合体的3D组织、机械转导和肌动蛋白束成核的位置 具体目标2：确定侧斑的3D组织、适应的位置以及可能的信号放大。 具体目标3：确定肌动蛋白核心的三维结构，包括肌动蛋白-肌动蛋白交联物和肌动蛋白束-膜连接物。 具体目标4：确定位于锥形立体纤毛区、牙根和角质板的分子机器的3D组织 我们将使用高压冷冻、冷冻替代和树脂包埋的毛束切片或冷冻水合的单个立体纤毛的电子断层扫描。束多蛋白复合体的3D结构将有助于从机械上理解我们的感觉或听力和平衡，并为未来对疾病组织的分析提供基线。

Our senses of hearing and balance rely on the proper development, function and maintenance of the hair bundle at the apical surface of inner ear hair cells. Hair bundles are composed of distinct multi-protein complexes of defined function and architecture that work together and that are responsible for mechanoelectrical transduction, adaptation and possibly signal amplification. Failure of function of any of these machineries leads to deafness or various degrees of hearing and balance impairments. Our goal is to visualize these stereocilia molecular machines in their native cellular context at molecular resolution. Our rationale is that the determination of the molecular 3D organization of these stereocilia complexes will ultimately lead to a mechanistic understanding of development, function and maintenance of this precision organelle. Specifically, we propose specifically to study four regions in stereocilia from lower (frog, zebrafish) and higher (mouse, guinea pig) vertebrates, each of which hosts a particular type of multi-protein complex, and plays a distinct role in hair bundle formation, function and/or maintenance. The four regions are 1.) the tip complex, 2.) the actin core, 3.) the adaptation complex, as well as 4.) the tapered region and the rootlets. Our specific aims are as follows: Specific Aim 1: Determine 3D organization of sterecilia tip complex, the site of mechanotransduction and actin bundle nucleation Specific Aim 2: Determine 3D organization of side plaque, the site of adaptation and possibly signal amplification. Specific Aim 3: Determine 3D organization of the actin core, including the actin-actin cross linkers and the actin bundle-membrane connectors. Specific Aim 4: Determine the 3D organization of molecular machines residing in the tapered stereocilia region, the rootlets and the cuticular plate We will employ electron tomography of either high-pressure frozen, freeze-substituted and resin-embedded hair bundle sections or of frozen-hydrated isolated individual stereocilia. The 3D architecture of the bundle multiprotein complexes will aid in a mechanistic understanding of our senses or hearing and balance and provide a baseline for future analysis of diseased tissue.