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

本补充请求的目标是获取台风 5 号以实现项目计划的实验 R01-EY026545，光感受器中的纤毛相关结构，以及 R01-EY031949 和其他的结构 贝勒医学院 NEI 资助的项目。这种仪器对于严格的 通过凝胶电泳分离的蛋白质、核酸、脂质和其他小分子的定量或 薄层色谱法。它将消除对过时的基于 X 射线胶片的检测的需要，并允许常规检测 免疫印迹的快速定量，这是我们 NEI 项目的主力技术。的简要总结 家长补助金如下。 该项目的目标是深入了解 p 的结构和分子基础 视杆细胞感觉纤毛的功能，并了解视杆细胞死亡的分子机制 纤毛病。有三个具体目标： 1. 使用冷冻电子断层扫描 (cryo-ET) 和最近的技术 确定纳米级三维结构的亚层析平均技术的发展 连接纤毛和基体的杆细胞重复结构的分辨率，包括微管 双联体和三联体、微管内部蛋白质、“Y 形链接”、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视网膜的杆状细胞 视网膜变性模型。 2. 使用超分辨率荧光来检验有关贩运的假设 特定蛋白质以及 IFT（鞭毛内运输）颗粒和 BBSome（一种形成包衣的物质）的作用 与致盲性纤毛病（Bardet-Biedl 综合征）有关的蛋白质复合物在视杆细胞中的纤毛运输中。二- 颜色超分辨率荧光和定量相互作用分析将用于评估假定的 IFT 蛋白或 BBS 蛋白与外段膜蛋白之间的相互作用，以及 通常从外节排除的蛋白质在 BBS 缺陷小鼠中错误地积累在那里。这些 实验将检验特定膜蛋白通过主动运输的假设 通过与 IFT 颗粒的结合来连接纤毛膜，而其他颗粒则通过 BBSome 会主动去除替代途径和排除的蛋白质。 3.使用小鼠模型进行测试 假设 CEP290 是从睫状体轴丝延伸的“Y 形链接”的主要组成部分 使用超分辨率荧光、传统 TEM 和冷冻电子断层扫描技术将 在不同发育阶段进行定时基因破坏或基因恢复，以区分起始与相反 与该蛋白相关的纤毛病病理生理学发展中的继发事件