Cilium-associated structures in rod cells

此补充请求的目标是获得第5号台风，以实现项目计划的实验 R01-EY026545，光感受器中的纤毛相关结构，以及R01-EY031949和其他 NEI资助的贝勒医学院项目。这类乐器对于严谨的 凝胶电泳法测定蛋白质、核酸、脂类及其他小分子的含量 薄层层析法。它将消除对过时的基于x射线胶片的检测的需要，并允许常规 快速定量免疫印迹，这是我们NEI项目的一项主要技术。简明地总结了 随后是家长赠款。 这个项目的目标是对p的结构和分子基础有一个彻底的了解。 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。它有三个具体目标：1.使用冷冻电子断层扫描(Cryo-ET)和最近的 亚层析平均法确定纳米级三维结构的研究进展 连接纤毛和基底体的杆状细胞重复结构的解析，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 是将硬件和软件的最新发展应用于野生视网膜和动物的视杆细胞 视网膜变性的模型。2.使用超分辨率荧光来验证关于贩运的假设 特定的蛋白质和关于IFT(鞭毛内转运)颗粒和BBSome(一种衣物形成)的作用 眼球纤毛运输中与致盲睫毛病变有关的蛋白质复合体(Bardet-Biedl综合征)。两个- 将使用彩色超分辨率荧光和定量相互作用分析来评估假设 IFT蛋白或BBS蛋白与外膜蛋白的相互作用 蛋白质通常被排除在BBS缺陷小鼠错误积累的外部片段之外。这些 实验将检验这种假设，即特定的膜蛋白通过 通过与IFT颗粒的结合连接纤毛膜，而其他的则通过 替代途径和被排除的蛋白质被BBSome主动移除。3.使用鼠标模型进行测试 CEP290是从睫状轴突伸出的“Y”形链的主要成分的假说 使用超分辨荧光、常规的透射电子显微镜和冷冻电子断层扫描 不同发育阶段的定时基因中断或基因恢复，以区分启动和相对启动 在与该蛋白相关的纤毛疾病的病理生理学发展过程中的继发性事件

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