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

本补充申请的目标是获得一架台风5号，以便进行项目计划的实验。 R 01-EY 026545，光感受器中的纤毛相关结构，以及R 01-EY 031949和其他 贝勒医学院的NEI资助项目。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X射线胶片检测的需要， 免疫印迹的快速定量，这是我们NEI项目的主力技术。一个简明的摘要， 家长补助金紧随其后。 这个项目的目标是发展一个彻底的了解的结构和分子基础的p 视杆感觉纤毛的功能，并了解视杆细胞死亡的分子机制 纤毛病变具体目标有三个：1。使用冷冻电子断层扫描（cryo-ET）和最近的 在确定纳米级三维结构的子断层图像平均化方面的进展 连接纤毛和基体的杆状细胞的重复结构的解析，包括微管 双联体和三联体、微管内蛋白、“Y形连接”、过渡纤维和附件。我们的目标 将最新硬件和软件发展应用于野生型视网膜和动物视网膜中的视杆细胞 视网膜变性的模型。2.使用超分辨率荧光来测试关于 特定的蛋白质和关于IFT（鞭毛内运输）颗粒和BBSome（一种形成涂层的蛋白质）的作用。 与致盲性睫状体病、Bardet-Biedl综合征有关的蛋白质复合物）在杆状体中的睫状体运输中的作用。二... 颜色超分辨荧光和定量相互作用分析将用于评估推定的 IFT蛋白或BBS蛋白与外节膜蛋白之间的相互作用，以及 在BBS缺陷小鼠中，通常从外节排除的蛋白质在那里错误积累。这些 实验将测试特定的膜蛋白通过膜被积极运输的假设。 通过与IFT颗粒的结合连接纤毛膜，而其他则通过 替代途径和排除的蛋白质被BBSome主动去除。3.使用小鼠模型进行测试 假设CEP 290是从纤毛轴丝延伸的“Y形连接”的主要成分 使用超分辨率荧光，常规TEM和冷冻电子断层扫描， 在不同的发育阶段定时基因破坏或基因恢复，以区分启动与 与这种蛋白质相关的纤毛病的病理生理学发展中的继发事件