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和锥形感受器使用其外部段检测光，高度专业 原发性纤毛的形式。整个体内的原发性睫状在感测细胞方面起着重要作用 其分子成分中的环境和遗传缺失（称为纤毛病）导致毁灭性 先天性疾病，包括盲目的残留变性。该项目的目的是开发 对原代纤毛功能的结构和分子基础的理解，重点是 感觉纤毛，并了解纤毛病中杆细胞死亡的分子机制。我们有 使用荧光和电子产品开发和应用创新的分子尺度成像方法 显微镜检查了这个问题，现在提出提出对成像技术进行其他改进 并使用它们来测试有关正常睫状结构和机制的假设，以及 视网膜纤毛病变动物模型中的病理生理机制。具体目的：1。使用冷冻电子 层析成像（冷冻-ET）和亚三图的最新发展平均，以确定三个 尺寸结构到连接纤毛的杆电池的重复结构的纳米分辨率 基本主体，包括微管双打和三胞胎，微管内蛋白，“ Y形链路”，过渡 纤维和附件。我们的目标是将硬件和软件的最新发展应用于两个 野生型视网膜和视网膜变性的动物模型。 2。使用上分辨率荧光进行测试 关于特定蛋白质的运输和IFT（flagallar transpers）颗粒的作用的假设 和BBSOME（在盲纤毛病，Bardet-Biedl综合征中实现的涂层蛋白质复合物） 在胶带中纤毛贩运。两色彩色超级分辨率荧光和定量相互作用分析将是 用于评估IFT蛋白或BBS蛋白与外部段膜之间的推定相互作用 蛋白质以及通常在外部段中排除的蛋白质，这些蛋白质在此中误解了 BBS缺陷小鼠。这些实验将检验以下假设：特定的膜蛋白是积极的 通过与IFT颗粒的关联，通过连接纤毛膜进行贩运，而其他 通过替代路线运输，排除的蛋白质被BBSOME积极去除。 3。使用 鼠标模型测试CEP290是“ Y形链接”的主要组成部分的假设 从睫状轴突到膜，使用上分辨率荧光，常规TEM和冷冻 电子断层扫描具有定时基因破坏或在不同发育阶段的基因恢复到 与纤毛病理生理学发展中的次要事件相反，启动起始 与该蛋白有关