Cilium-associated structures in rod cells

杆状细胞中的纤毛相关结构

• 批准号: 10303692
• 负责人: THEODORE G WENSEL
• 金额: $ 15.99万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10303692
• 关键词: Animal Model; Bardet-Biedl Syndrome; Carrier Proteins; Cell Death; Cilia; Color; Computer software; Cryo-electron tomography; Deficiency Diseases; Detection; Development; Equipment; Event; Eye diseases; Fiber; Fluorescence; Functional disorder; Funding; Genes; Genetic; Goals; Hereditary Disease; Image; Lead; Link; Lipids; Medicine; Membrane; Membrane Proteins; Microtubules; Molecular; Mus; Nerve Degeneration; Nucleic Acids; Pathology; Photoreceptors; Proteins; Retina; Retinal Degeneration; Rod; Role; Route; Secondary to; Sensory; Structure; Techniques; Testing; Thin Layer Chromatography; Tissues; Tomogram; Triplet Multiple Birth; X-Ray Film; appendage; base; ciliopathy; college; experimental study; gel electrophoresis; instrument; kinetosome; mouse model; nanometer resolution; novel therapeutic intervention; parent grant; particle; protein complex; restoration; retinal rods; small molecule; three dimensional structure; trafficking

The goal of this Supplement request is to acquire a Typhoon 5 to enable the experiments planned for project R01-EY026545, Cilium-Associated Structures in Photoreceptors, as well as those for R01-EY031949 and other NEI-funded projects at Baylor College of Medicine. An instrument of this kind is essential for rigorous quantification of proteins, nucleic acids, lipids and other small molecules separated by gel electrophoresis or thin-layer chromatography. It will eliminate the need for outmoded x-ray film-based detection and allow routine rapid quantification of immunoblots, a workhorse technique for our NEI projects. A condensed summary of the parent grant follows. The goal of this project is to develop a thorough understanding of the structural and molecular basis of p function of the rod sensory cilium, and to understand the molecular mechanisms of rod cell death in ciliopathies. There are three Specific Aims: 1. Use cryo-electron tomography (cryo-ET) and recent developments in sub-tomogram averaging to determine the three-dimensional structure to nanometer resolution of repeating structures of the rod cell connecting cilium and basal body, including microtubule doublets and triplets, microtubule inner proteins, “Y-shaped links”, transition fibers and appendages. Our goal is to apply recent developments in hardware and software to rod cells in both wild type retinas and in animal models of retinal degeneration. 2. Use superresolution fluorescence to test hypotheses about trafficking of specific proteins and about the roles of IFT (intraflagellar transport) particles and the BBSome (a coat-forming protein complex implicated in the blinding ciliopathy, Bardet-Biedl syndrome) in ciliary trafficking in rods. Two- color superresolution fluorescence and quantitative interaction analysis will be used to assess putative interactions between IFT proteins or BBS proteins and outer segment membrane proteins, as well as well as proteins normally excluded from the outer segment which mis-accumulate there in BBS-deficient mice. These experiments will test the hypothesis that specific membrane proteins are actively trafficked through the connecting cilium membrane through their association with IFT particles, whereas others are transported via alternative routes and excluded proteins are actively removed by the BBSome. 3. Use mouse models to test the hypotheses that CEP290 is a major component of the “Y-shaped links” extending from the ciliary axoneme to the membrane, using superresolution fluorescence, conventional TEM, and cryo-electron tomography with timed gene disruption or gene restoration at different developmental stages to distinguish initiating as opposed to secondary events in the development of the pathophysiology of ciliopathies associated with this protein

此补充要求的目的是获得台风5，以启用计划的实验 R01-ey026545，感光器中与纤毛相关的结构，以及R01-EY031949和其他的结构 贝勒医学院的Nei资助项目。这种工具对于严格 定量蛋白质，核酸，脂质和其他小分子，这些分子被凝胶电泳或 薄层色谱。它将消除对基于X射线膜的过时检测的需求，并允许例行 免疫印迹的快速量化，这是我们的NEI项目的主力技术。凝结的摘要 父母赠款如下。 该项目的目的是对P的结构和分子基础进行彻底理解 杆感觉纤毛的功能，并了解杆细胞死亡的分子机制 纤毛病。有三个特定的目的：1。使用低温电子层析成像（Cryo-ET）和最近 亚三图中的发展，以确定三维结构到纳米的结构 连接纤毛和基本体的杆电池重复结构的分辨率，包括微管 双重和三重态，微管内蛋白，“ Y形链路”，过渡纤维和附属物。我们的目标 是将硬件和软件的最新发展应用于野生型视网膜和动物的杆细胞 视网膜变性模型。 2。使用上分辨率荧光来检验有关贩运的假设 特定的蛋白质以及IFT（flagellar运输）颗粒和BBSOME（涂层形成）的作用 蛋白质复合物在纤毛的纤毛病，bardet-biedl综合征中隐含在杆状贩运中。二- 颜色超级分辨率荧光和定量相互作用分析将用于评估推定 IFT蛋白或BBS蛋白与外部节膜蛋白之间的相互作用以及 蛋白质通常排除在外部片段中，这些蛋白质在BBS缺陷型小鼠中误解。这些 实验将检验以下假设：特定的膜蛋白通过 通过与IFT颗粒的关联连接纤毛膜，而其他纤维膜是通过 BBSOME积极去除替代路线和排除的蛋白质。 3。使用鼠标模型进行测试 CEP290是从睫状轴突延伸的“ Y形链接”的主要组成部分的假设 使用上分辨率荧光，常规TEM和冷冻电子层析成像到膜上 定时基因破坏或在不同发育阶段的基因恢复，以区分起始 在与该蛋白质相关的纤毛病理生理学发展中的次要事件