Cilium-associated structures in rod cells

杆状细胞中的纤毛相关结构

• 批准号: 10601082
• 负责人: THEODORE G WENSEL
• 金额: $ 40万
• 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/10601082
• 关键词: Animal Model; Bardet-Biedl Syndrome; Carrier Proteins; Cell Death; Cellular Structures; Cilia; Color; Computer software; Cryo-electron tomography; Deficiency Diseases; Development; Disease; Electron Microscopy; Electrons; Environment; Event; Exclusion; Eye diseases; Fiber; Fluorescence; Fluorescence Microscopy; Functional disorder; Genes; Genetic; Genetic Techniques; Goals; Hereditary Disease; Imaging technology; Lead; Light; Link; Membrane; Membrane Proteins; Microtubules; Molecular; Mus; Nerve Degeneration; Noise; Pathology; Pharmaceutical Preparations; Play; Proteins; Resolution; Retina; Retinal Degeneration; Retinal Photoreceptors; Rod; Role; Route; Secondary to; Sensory; Shapes; Signal Transduction; Structure; Testing; Tissues; Triplet Multiple Birth; Vertebrate Photoreceptors; appendage; ciliopathy; experimental study; imaging approach; improved; innovation; kinetosome; molecular scale; mouse model; nanometer resolution; novel; novel therapeutic intervention; particle; protein complex; protein function; restoration; retinal rods; three dimensional structure; trafficking; ultra high resolution

Rod and cone photoreceptors of the vertebrate retina detect light using their outer segments, highly specialized forms of primary cilia. Primary ciliary throughout the body play important roles in sensing the cellular environment, and genetic deletions in their molecular components, known as ciliopathies, lead to devastating congenital diseases, including blinding forms of retinal degeneration. The goal of this project is to develop a thorough understanding of the structural and molecular basis of primary cilium function, with a focus on the rod sensory cilium, and to understand the molecular mechanisms of rod cell death in ciliopathies. We have developed and applied innovative molecular-scale imaging approaches using fluorescence and electron microscopy to this problem, and now propose to introduce additional improvements in the imaging technology and to use them to test hypotheses about normal ciliary structures and mechanisms, and about pathophysiological mechanisms in animal models of retinal ciliopathies. 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

脊椎动物视网膜的杆状和锥状光感受器利用其高度专业化的外节检测光 初级纤毛的形式。遍布全身的初级纤毛在感知细胞信号方面发挥着重要作用 环境及其分子成分的基因缺失（称为纤毛病）会导致毁灭性的 先天性疾病，包括致盲性视网膜变性。该项目的目标是开发一个 彻底了解初级纤毛功能的结构和分子基础，重点是杆 感觉纤毛，并了解纤毛病中杆状细胞死亡的分子机制。我们有 开发并应用了利用荧光和电子的创新分子尺度成像方法 显微镜解决了这个问题，现在建议在成像技术上引入额外的改进 并用它们来检验关于正常睫状体结构和机制的假设，以及关于 视网膜纤毛病动物模型的病理生理机制。具体目标： 1. 使用低温电子 断层扫描（冷冻电子断层扫描）和亚断层扫描平均的最新发展，以确定三 连接纤毛和杆状细胞的重复结构的尺寸结构到纳米分辨率 基体，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杆细胞 野生型视网膜和视网膜变性动物模型。 2. 使用超分辨荧光进行测试 关于特定蛋白质运输和 IFT（鞭毛内运输）颗粒作用的假设 和 BBSome（一种与致盲性纤毛病、Bardet-Biedl 综合征有关的外壳形成蛋白复合物） 视杆细胞的纤毛贩运。双色超分辨荧光和定量相互作用分析将 用于评估 IFT 蛋白或 BBS 蛋白与外节膜之间的假定相互作用 蛋白质，以及通常被排除在外段之外的蛋白质，这些蛋白质错误地积累在其中 BBS 缺陷小鼠。这些实验将检验特定膜蛋白活跃的假设 通过与 IFT 颗粒的结合，通过连接纤毛膜进行运输，而其他的 通过替代途径运输，排除的蛋白质被 BBSome 主动去除。 3. 使用 小鼠模型来测试 CEP290 是“Y 形链接”延伸的主要组成部分的假设 使用超分辨率荧光、传统 TEM 和冷冻技术，从纤毛轴丝到膜 在不同发育阶段进行定时基因破坏或基因恢复的电子断层扫描 区分纤毛病病理生理学发展中的始发事件和继发事件 与该蛋白质相关