权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Supplement for childcare costs for NRSA fellow

NRSA 研究员的儿童保育费用补助

基本信息

批准号：
10394667
负责人：
Sarah Clark
金额：
$ 0.25万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-01 至 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10394667
关键词：
3-Dimensional Affinity Antibodies Antibody Affinity Antigens Architecture Audiology Biological CDH23 gene Cells Cochlea Complex Cryo-electron tomography Cryoelectron Microscopy Data Collection Development Disease Electrons Electrophysiology (science)Elements Environment Esthesia Extracellular Protein Filament Future Goals HMGA2 gene Hair Hair Cells Hearing Hearing problem Ice Image Individual Inner Hair Cells Knowledge Label Labyrinth Link Lipoma Liquid substance Maps Measures Mechanics Membrane Proteins Methodology Methods Modeling Molecular Monoclonal Antibodies Movement Multiple Partners Mus Mutation National Research Service Awards Organism PCDH15 gene Photobleaching Play Production Proteins Quantum Dots Research Resolution Role Signal Transduction Site Stimulus Structure Techniques Therapeutic Tissues Tomogram Touch sensation Usher Syndrome Type 1 Visualization Work antibody conjugate cost deaf design experimental study fluorophore hereditary hearing loss insight link protein mechanotransduction novel therapeutics reconstruction single molecule sound stoichiometry structural biology three dimensional structure tomography treatment strategy

项目摘要

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.

项目摘要位于内耳毛束尖端的机械传导机制负责我们的运动和声音的感觉。每束由约100根静纤毛组成，呈阶梯状排列由原钙粘蛋白15（PCDH 15）和钙粘蛋白23（CDH 23）。通过声音和流体运动的发束的偏转导致发束的打开。机械转导通道（MEC）复合物位于较低的尖端链接插入位点，导致电信号了除了尖端连接蛋白外，MEC复合物还有三个假定的组成部分： HMGIC融合伴侣样5蛋白LHFPL 5（也称为TMHS），跨膜内耳蛋白TMIE，和跨膜样通道蛋白TMC 1/2，它们可能是复合物的成孔亚基。虽然已知这些蛋白质相互作用形成听觉所必需的机械转导复合物，该络合物的精确组成、化学计量和结构仍然是难以捉摸的。这项提案的目的是是阐明的组成和结构的MEC复杂使用单分子技术和冷冻，电子断层摄影术所提出的工作的主要挑战是MEC复合物在天然组织我们已经开发了针对四种MEC复合物组分中的三种的高亲和力抗体这将是我们解决这一问题的方法的关键要素。在第一个目标中，我将利用这些抗体用单分子下拉法从小鼠耳蜗毛细胞中提取并检测MEC复合物以评估MEC复合物的组成。光漂白和单分子定量实验将允许以确定MEC复合物的化学计量并测量每个耳蜗的MEC复合物的数量。在第二个目标，这个建议，我将阐明的MEC复杂的结构，使用冷冻电子断层扫描。我已经开发了一种方法来制备冷冻电镜网格与静纤毛在玻璃冰，并获得了有前途的初步断层扫描我将用我们的抗体结合量子点来标记低温下的MEC复合物- EM网格和确定复杂的3D结构。阐明MEC的架构和组成复合物将提供有价值的洞察难以捉摸的机制毛细胞mechanotransductions和开放为聋人开发新的治疗方法和治疗策略打开了大门。