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: 10334877
• 负责人: Michael Robichaux
• 金额: $ 29.49万
• 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/10334877
• 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; Precipitation; Proteins; Research; Resolution; 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; trafficking; 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）：哺乳动物视杆细胞光感受器中视紫红质运输的分子尺度绘图， Robichaux，PL 项目总结/摘要 在脊椎动物视网膜的视杆细胞感光神经元中，视紫红质（Rho）是感光的G蛋白， 一种偶联受体，在专门的感觉纤毛内密集地包装成膜盘堆叠 也就是所谓的外节杆状细胞不断更新这些细胞盘，因此大量的Rho蛋白 必须不断地合成并运输到每个杆中的外部节段。为了保持这种沉重的 在Rho的生物合成过程中，杆具有高度调节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非常重要的任务，当它被破坏时，它会导致致盲性视网膜疾病， 视网膜色素变性最近的证据表明，在哺乳动物杆光感受器交替，非传统 然而，这些途径仍然未被发现。另一个未充分研究的组件 Rho在棒中的运输是被称为基体的基本结构航点，其位于 杆的生物合成区和细连接纤毛桥之间的临界边界， 外段基体由两个中心粒组成，中心粒具有相关的子结构，包括 中心粒卫星和远端附属物。这些都是Rho相互作用和对接的潜在位点， 运输到外节;然而，这些结构也没有在哺乳动物中进行严格的研究。 rods日期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体中，并且内质网（ER）膜可以 也位于基体上。在本提案的第一个目的中，我们将首先测试新生Rho蛋白是否被 在哺乳动物视杆细胞的基体区域内的局部内质网膜中合成，作为潜在的 替代Rho运输途径。我们将产生一种新型的ER基因敲入融合小鼠来进行 全面的，超分辨率映射的整个本地ER网络杆。我们将定量地映射到 这个ER网络是Rho蛋白、核糖体（作为粗糙ER）和Rho mRNA作为表达的位点的精确定位。 本地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 在杆基体内的局部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在中心粒卫星和远端附属物处停靠在基体上， 蛋白质与这两个基体子结构的组分蛋白质的相互作用。我们将执行一个 严格的纳米级映射这些结构在小鼠杆，其次是定量测试的Rho co- 定位与卫星和远端附属物的组成蛋白。我们会证实这些 与生化共沉淀测定的新相互作用。最后，我们将进行一种新的，有针对性的抑制， 通过视网膜下递送携带机器的病毒载体， 破坏了小鼠杆状细胞的这些结构。通过这些实验，我们将确定这些基体的作用 Rho运输和视杆光感受器稳态中的结构。