Cilium-associated structures in rod cells

脊椎动物视网膜的视杆和视锥感光器利用它们的外节探测光线，高度专门化 初生纤毛的形式。遍及全身的初级纤毛在感知细胞方面起着重要作用 环境及其分子成分的遗传缺失，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。这个项目的目标是开发一种 深入了解初级纤毛功能的结构和分子基础，重点是纤毛杆部 纤毛感觉，并了解纤毛疾病中杆状细胞死亡的分子机制。我们有 利用荧光和电子开发和应用创新的分子尺度成像方法 显微镜解决了这个问题，现在建议在成像技术方面引入额外的改进 并用它们来检验关于正常纤毛结构和机制的假说，以及关于 视网膜纤毛病变动物模型的病理生理机制。具体目标：1.使用低温电子 体层摄影术(冷冻-ET)和亚断层图像平均以确定三个- 连接纤毛和纤毛的杆状细胞重复结构的空间结构到纳米分辨率 基底体包括微管二联体和三联体，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方面的最新发展应用于这两种情况下的杆状电池 野生型视网膜和视网膜变性的动物模型。2.使用超分辨荧光进行检测 关于特定蛋白质运输和IFT(鞭毛内转运)颗粒作用的假设 和BBSome(一种与Bardet-Biedl综合征致盲睫毛有关的外套形成蛋白复合体) 在纤毛交易中的杆状。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蛋白与外节膜之间可能的相互作用 蛋白质，以及通常从外层片段排除的蛋白质，这些蛋白质在那里错误地积累 BBS缺陷小鼠。这些实验将检验特定的膜蛋白活跃的假说。 通过它们与IFT颗粒的结合通过连接的纤毛膜运输，而其他的 通过替代途径运输，被排除的蛋白质被BBSome主动移除。3.使用 用小鼠模型检验CEP290是“Y形连接”延伸的主要成分的假说 从纤毛轴丝到膜，使用超分辨荧光、常规透射电子显微镜和冷冻- 在不同发育阶段进行定时基因中断或基因修复的电子断层扫描 区分纤毛疾病病理生理学发展中的始发事件和继发事件 与这种蛋白质有关

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