Hair Bundle Structure and Dynamics

发束结构和动力学

基本信息

批准号：
9296117
负责人：
Peter Gordon Barr-Gillespie
金额：
$ 42.65万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-10 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9296117
关键词：
Affect Alleles Antibodies Biological Assay Biological Models Biological Process Cells Clustered Regularly Interspaced Short Palindromic Repeats Development Electron Microscopy Epithelium Equilibrium Fluorescence-Activated Cell Sorting Functional disorder Funding Gene Deletion Gene Expression Genes Genetic Goals Hair Hair Cells Health Hearing Heart Hereditary Disease Human Immunofluorescence Immunologic Individual Knock-out Knockout Mice Laboratories Lighting Location Mass Spectrum Analysis Measures Mechanics Methods Microfluidics Microscopy Molecular Genetics Monitor Mus Nature Organelles Pathway interactions Phosphorylation Play Post-Translational Protein Processing Proteins Proteome Proteomics Quality of life Regulation Resolution Reverse Transcriptase Polymerase Chain Reaction Role Sensory Signal Transduction Stereocilium Structure Supporting Cell System Techniques Technology Testing Time Transcript Ubiquitination Utricle structure biological systems deafness electron tomography experimental study hearing impairment insight knockout gene light microscopy mechanotransduction mouse model mutant neuronal cell body novel protein expression public health relevance single molecule sound therapy design tool transcription factor vibration

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this laboratory is reveal the structure of the hair cell's mechanosensitive organelle, the hair bundle, and to determine how structural features of the bundle are responsible for its mechanotransduction function. In this proposal, we extend on the accomplishments of the previous funding period-where we developed a parts list for hair bundles-and here determine the sequence in which bundle proteins are expressed during development, as well as the mechanisms that control their expression and function. In Aim 1, we focus on transcripts for ~175 key bundle proteins, using multiplex RT-qPCR assays to quantify their dynamics in whole sensory epithelium and in pools of isolated hair cells. We also examine the same transcript in isolated single cells, allowing a more detailed understanding of the timing and stochastic nature of gene expression. In Aim 2, we quantify many or most of these proteins by targeted proteomics assays, and also determine their time course for transfer from the soma to the bundle. In Aim 3, we focus on structure; using cryo-electron tomography we will gain a fine understanding of stereocilium structure, while we will use immunofluorescence and structured illumination microscopy to determine which proteins target to which structures. Using conventional conditional alleles or CRISPR/Cas9 knockouts, experiments in both Aims 1 and 3 will test the roles of specific transcription factors and bundle proteins in, respectively, control of bundle transcript expression and bundle structure. We anticipate that some of the bundle proteins we are examining will be affected in genetic diseases that cause hearing impairment.

描述（由申请人提供）：该实验室的长期目标是揭示毛细胞的机械敏感细胞器（发束）的结构，并确定毛束的结构特征如何影响其机械转导功能。我们延续了上一个资助阶段的成就——我们开发了发束的零件清单——并在此确定了发束蛋白在发育过程中表达的序列，以及控制其表达和功能的机制。如图 1 所示，我们专注于约 175 个关键束蛋白的转录本，使用多重 RT-qPCR 测定来量化它们在整个感觉上皮和分离的毛细胞池中的动态。我们还检查了分离的单细胞中的相同转录本，从而获得更详细的信息。在目标 2 中，我们通过靶向蛋白质组学分析对许多或大部分蛋白质进行了定量，并确定了它们从体细胞转移到细胞束的时间过程。 3，我们专注于结构；使用冷冻电子断层扫描我们将深入了解静纤毛结构，同时我们将使用免疫荧光和结构照明显微镜来确定哪些蛋白质靶向哪些结构。目标 1 和 3 中的实验将分别测试特定转录因子和束蛋白在控制束转录表达和束结构中的作用。患有导致听力障碍的遗传性疾病。