Molecular -Scale Mapping of Rhodopsin Trafficking in Mammalian Rod Photoreceptors

哺乳动物视杆光感受器中视紫红质运输的分子尺度图谱

• 批准号: 10593138
• 负责人: Michael Robichaux
• 金额: $ 29.05万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-20 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10593138
• 关键词: Anabolism; Binding; Biochemical; Biological Assay; Biology; Body Regions; CRISPR/Cas technology; Cell membrane; Cells; Cellular biology; Centers of Research Excellence; Centrioles; Cilia; Color; Communities; Complement; Data; Dependovirus; Disease; Distal; Docking; Electron Microscopy; Endoplasmic Reticulum; Environment; Event; Fluorescence; Fluorescence Microscopy; Fostering; G-Protein-Coupled Receptors; Genetic; Genetic Models; Golgi Apparatus; Homeostasis; Imaging Device; Knock-in; Knock-in Mouse; Light; Link; Location; Maps; Mediating; Membrane; Mentors; Messenger RNA; Methods; Microscopy; Molecular; Movement; Mus; Mutation; Neurons; Pathogenicity; Pathway interactions; Photons; Photoreceptors; Phototransduction; Proteins; Research; Retina; Retinal Diseases; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Ribosomes; Rod; Role; Rough endoplasmic reticulum; Sensory; Site; Structure; System; Techniques; Testing; Thinness; Time; Translations; Viral Vector; Vision; Vision research; Visual Signal Transduction Pathway; appendage; base; ciliopathy; early onset; experimental study; imaging approach; interest; kinetosome; molecular pathology; molecular scale; mouse genetics; mouse model; nanoscale; novel; photoreceptor cell inner segment; programs; protein protein interaction; retinal neuron; retinal rods; superresolution microscopy; trafficking; ultra high resolution; vision science

Project 001 (273): Molecular -Scale Mapping of Rhodopsin Trafficking in Mammalian Rod Photoreceptors, Robichaux, PL PROJECT SUMMARY/ABSTRACT In rod photoreceptor neurons of the vertebrate retina, rhodopsin (Rho) is the light-sensing, G protein- coupled receptor that is densely packaged into stacks of membrane discs within a specialized sensory cilium known as the outer segment. Rods continuously renew these discs, and so an enormous amount of Rho protein must be constantly synthesized and trafficked to the outer segment in each rod. To maintain this heavy biosynthesis of Rho, rods feature a highly regulated yet ill-characterized secretory and trafficking network. Rho trafficking in rod neurons is such an essential task that, when disrupted, it causes blinding retinal diseases such as retinitis pigmentosa. Recent evidence in mammalian rod photoreceptors suggest alternate, unconventional pathways for Rho trafficking; however, these pathways remain undiscovered. Another understudied component of Rho trafficking in rods is the essential structural waypoint known as the basal body, which is located at the critical boundary between the biosynthetic zone of rods and the thin connecting cilium bridge that leads to the outer segment. The basal body is composed of two centrioles that have associated sub-structures, including the centriolar satellites and distal appendages. These are each potential sites of Rho interaction and docking before transport into the outer segment; however, these structures have also not been rigorously studied in mammalian rods to date. Our preliminary data using advanced, super-resolution microscopy methods indicate that Rho molecules are densely accumulated in the basal body, and that endoplasmic reticulum (ER) membranes may also be located at the basal body. In the first aim of this proposal, we will first test if nascent Rho protein is being synthesized in local ER membranes within the basal body region of mammalian rods as part of a potential alternate Rho trafficking pathway. We will generate a novel knock-in ER fusion mouse to perform a comprehensive, super-resolution mapping of the entire local ER network in rods. We will quantitatively map onto this ER network the precise localization of Rho proteins, ribosomes (as rough ER), and Rho mRNA as sites for local Rho translation. Collectively, these experiments will define new Rho trafficking events and establish a new local ER network within the rod basal body. In our second aim, we will use complimentary approaches to test if Rho proteins dock onto the basal body at the centriolar satellites and distal appendages via specific protein- protein interactions with the component proteins of those two basal body sub-structures. We will perform a rigorous nanoscale mapping of these structures in mouse rods, followed by a quantitative test of Rho co- localization with the component proteins of the satellites and the distal appendages. We will corroborate these new interactions with biochemical co-precipitation assays. Finally, we will perform a novel, targeted inhibition of the satellites and distal appendages, by subretinal delivery of viral vectors carrying machinery to genetically disrupt these structures in mouse rods. With these experiments, we will establish the role of these basal body structures in Rho trafficking and rod photoreceptor homeostasis.

项目001(273)：视紫红质在哺乳动物视杆感受器中运输的分子尺度图谱， 罗比肖，PL 项目摘要/摘要 在脊椎动物视网膜的视杆状感光神经元中，视紫红质(Rho)是感光的G蛋白。 偶联受体，在特殊的感觉纤毛内被密集地包装成成堆的膜盘 也就是所谓的外段。杆不断地更新这些圆盘，因此大量的Rho蛋白质 必须不断地合成并运输到每根棒的外部部分。为了保持这份沉重 Rho的生物合成，杆的特点是一个高度调控但特征不佳的分泌和运输网络。Rho 视杆神经元的运输是一项如此重要的任务，当被破坏时，它会导致视网膜失明疾病，如 是视网膜色素变性。哺乳动物视杆感光器的最新证据表明，非传统的 Rho贩运的途径；然而，这些途径仍然未被发现。另一个未被研究的组成部分 Rho棒运输的关键结构点是被称为基体部的，它位于 杆状生物合成区和纤毛桥之间的临界边界，纤毛桥通向 外部管段。基本体由两个中心粒组成，这两个中心粒具有相关联的亚结构，包括 中心粒卫星和远端附属物。这些都是以前Rho交互和对接的潜在地点 转运到外节；然而，这些结构在哺乳动物中也没有得到严格的研究。 到目前为止是棒子。我们使用先进的超分辨率显微镜方法的初步数据表明，Rho 分子密集地聚集在基底体，内质网(ER)膜可能 也位于基体部。在这项提案的第一个目标中，我们将首先测试新生Rho蛋白是否正在 在哺乳动物杆基体区的局部ER膜中合成，作为潜在的一部分 另一条Rho运输路径。我们将产生一种新型的敲入内质网融合小鼠来执行 在RODS中对整个本地ER网络进行全面、超分辨率的测绘。我们将定量测绘到 这个ER网络将Rho蛋白、核糖体(作为粗略的ER)和Rho mRNA的精确定位作为 当地RHO翻译。总的来说，这些实验将定义新的RHO贩运事件，并建立新的 视杆基底体内的局部ER网络。在我们的第二个目标中，我们将使用互补的方法来测试 Rho蛋白通过特定的蛋白质对接到位于中心粒卫星和远端附件的基底上。 蛋白质与这两个基础体亚结构的组成蛋白质相互作用。我们将表演一场 在老鼠棒中对这些结构进行了严格的纳米级映射，随后对Rho co. 利用卫星和远端附属物的组成蛋白进行定位。我们将证实这些 与生化共沉淀分析的新相互作用。最后，我们将执行一项新颖的、有针对性的抑制 卫星和远端附件，通过视网膜下运送携带基因机器的病毒载体 破坏老鼠棒中的这些结构。通过这些实验，我们将确定这些基础体的作用 Rho转运和杆状感光细胞动态平衡中的结构。