Project B

项目B

基本信息

批准号：
8377329
负责人：
MANFRED AUER
金额：
$ 39.14万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8377329
关键词：
Actins Address Animals Ankle Apical Architecture Beds Biological Biological Models Biological Preservation Cavia Cell membrane Comparative Study Complex Data Development Dimensions Distal Electron Microscopy Epithelium Equilibrium Failure Filament Fimbrin Freezing Future Genes Genetic Goals Hair Hair Cells Head Hearing In Vitro Individual Insecta Label Labyrinth Lead Link Maintenance Maps Membrane Microfilaments Modeling Molecular Molecular Machines Molecular Motors Morphologic artifacts Motor Multiprotein Complexes Mus Muscle Mutation Myosin ATPase Nature Organelles Pattern Physiology Plant Resins Play Positioning Attribute Preparation Process Proteins Rana Rana catesbeiana Resolution Role Sampling Sensory Shapes Side Signal Transduction Site Speed Staining method Stains Stereocilium Stimulus Structure Surface Syndrome Thick Filament Tissues Tomogram Vertebrates Work Zebrafish base cold temperature crosslink deafness density depolymerization electron tomography equilibration disorder hearing impairment human CDH23 protein in vitro Model in vivo interest macromolecule monomer pressure protein complex reconstruction spatial relationship species difference three-dimensional modeling tomography

项目摘要

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.

我们的听觉和平衡感依赖于内耳毛细胞顶端表面毛束的正常发育、功能和维护。发束由具有明确功能和结构的不同多蛋白复合物组成，它们协同工作，负责机电转导、适应和可能的信号放大。这些机器的任何功能故障都会导致耳聋或不同程度的听力和平衡障碍。我们的目标是以分子分辨率在其天然细胞环境中可视化这些静纤毛分子机器。我们的理由是，确定这些静纤毛复合物的分子 3D 组织将最终导致对这种精密细胞器的发育、功能和维护的机械理解。具体来说，我们建议专门研究低等（青蛙、斑马鱼）和高等（小鼠、豚鼠）脊椎动物的静纤毛的四个区域，每个区域都承载特定类型的多蛋白复合物，并在发束形成中发挥独特的作用、功能和/或维护。这四个区域是 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 sterecilia 尖端复合体的 3D 组织、机械传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、适应位点以及可能的信号放大。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束膜连接器。具体目标 4：确定锥形静纤毛区域、细根和角质板中分子机器的 3D 组织我们将采用电子断层扫描技术对高压冷冻、冷冻替代和树脂嵌入的发束切片或冷冻水合的分离的单个静纤毛进行电子断层扫描。束多蛋白复合物的 3D 架构将有助于我们对感官或听觉和平衡的机械理解，并为未来分析患病组织提供基线。