Cilium-associated structures in rod cells

杆状细胞中的纤毛相关结构

• 批准号: 10382249
• 负责人: THEODORE G WENSEL
• 金额: $ 38.8万
• 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/10382249
• 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; 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 Cone; Retinal Degeneration; Retinal Photoreceptors; Rod; Role; Route; Secondary to; Sensory; Signal Transduction; Structure; Testing; Tissues; Tomogram; Triplet Multiple Birth; Vertebrate Photoreceptors; appendage; ciliopathy; experimental study; imaging approach; innovation; kinetosome; molecular scale; mouse model; nanometer resolution; novel; novel therapeutic intervention; particle; protein complex; protein function; restoration; retinal rods; three dimensional structure; trafficking

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

脊椎动物视网膜的视杆和视锥光感受器利用它们的外节来检测光， 初级纤毛的形式。遍布全身的初级纤毛在感受细胞的 环境，和基因缺失，在其分子组成部分，被称为纤毛病，导致毁灭性的 先天性疾病，包括致盲形式的视网膜变性。该项目的目标是开发一个 深入了解初级纤毛功能的结构和分子基础，重点是杆 感觉纤毛，并了解纤毛疾病中杆细胞死亡的分子机制。我们有 开发和应用创新的分子尺度成像方法，使用荧光和电子 显微镜来解决这个问题，现在建议在成像技术中引入额外的改进 并使用它们来测试关于正常纤毛结构和机制的假设，以及关于 视网膜纤毛病变动物模型的病理生理学机制。具体目标：1。使用低温电子 断层扫描（cryo-ET）和最近的发展，在子断层平均，以确定三个- 三维结构到纳米分辨率的杆状细胞连接纤毛的重复结构， 基体，包括微管双联体和三联体，微管内部蛋白，“Y形连接”，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两种细胞中的视杆细胞， 野生型视网膜和视网膜变性的动物模型。2.使用超分辨荧光检测 关于特定蛋白质运输和IFT（鞭毛内运输）颗粒作用的假说 和BBSome（一种与致盲性纤毛病，Bardet-Biedl综合征有关的涂层形成蛋白复合物） 睫状体的杆状物交易双色超分辨荧光和定量相互作用分析将是 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，以及通常从外节排除的蛋白质，这些蛋白质在外节中错误地积累。 BBS缺陷小鼠。这些实验将验证特定的膜蛋白在细胞膜上活跃的假设。 通过与IFT颗粒的结合，通过连接纤毛膜进行运输，而其他 通过替代途径转运，被排除的蛋白质被BBSome主动清除。3.使用 小鼠模型，以测试假设CEP 290是“Y形链接”延伸的主要组成部分， 使用超分辨率荧光、常规TEM和冷冻技术，从睫状体轴丝到细胞膜 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， 在纤毛病变的病理生理学发展中区分起始事件和继发事件 与这种蛋白质相关