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正常发育、功能和维护。毛束是由不同的多蛋白质复合物的定义功能和结构，一起工作，并负责机械电转导，适应和可能的信号放大。任何这些机器的功能故障导致耳聋或不同程度的听力和平衡障碍。 我们的目标是可视化这些静纤毛分子机器在他们的原生细胞背景下的分子分辨率。我们的基本原理是，这些静纤毛复合物的分子3D组织的确定将最终导致这种精密细胞器的发展，功能和维护的机械理解。具体而言，我们建议专门研究四个地区的静纤毛从较低的（青蛙，斑马鱼）和较高的（小鼠，豚鼠）脊椎动物，其中每一个主机一个特定类型的多蛋白复合物，并发挥独特的作用，毛束的形成，功能和/或维护。这四个区域是1）。尖端复合物，2.）肌动蛋白核心，3.）适应复杂，以及4.）锥形区域和小根。 我们的具体目标如下： 具体目标1：确定sterecilia尖端复合体的3D组织，机械转导和肌动蛋白束成核的位点 具体目标2：确定侧斑块的3D组织、适应部位和可能的信号放大。 具体目标3：确定肌动蛋白核心的3D组织，包括肌动蛋白-肌动蛋白交联剂和肌动蛋白链-膜连接器。 具体目标4：确定位于锥形静纤毛区域、小根和表皮板中的分子机器的3D组织 我们将采用电子断层扫描的高压冷冻，冷冻替代和树脂包埋的头发束部分或冷冻水合孤立的个人静纤毛。束多蛋白复合物的3D结构将有助于对我们的感官或听觉和平衡的机械理解，并为未来分析病变组织提供基线。

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.