The role of the photoreceptor tetraspanin Rds in outer segment morphogenesis

光感受器四跨膜蛋白 Rds 在外节形态发生中的作用

• 批准号: 8018107
• 负责人: Shannon M Conley
• 金额: $ 5.68万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8018107
• 关键词: A Mouse; ANXA2 gene; Accounting; Actins; Adhesions; Affinity; Affinity Chromatography; Apoptosis; Binding; Biochemical; Biological Models; Cadherins; Cattle; Cell Adhesion; Cell Adhesion Molecules; Cell Survival; Cell physiology; Cellular biology; Co-Immunoprecipitations; Complementary DNA; Complex; Confocal Microscopy; Coupled; Cytoskeleton; Data; Defect; Development; Disease; Electron Microscopy; Goals; Gold; Human; Immunoelectron Microscopy; Immunohistochemistry; Immunoprecipitation; Internet; Interruption; Knock-out; Knockout Mice; Label; Leucine Zippers; Link; Lipids; Macular degeneration; Maintenance; Membrane; Membrane Microdomains; Methods; Microtubules; Modeling; Morphogenesis; Mouse Strains; Mus; Mutation; National Eye Institute; Neural Retina; Outcome; Photoreceptors; Process; Proteins; Proteomics; Public Health; Radiolabeled; Regulation; Research; Research Personnel; Retina; Retinal; Retinal Cone; Retinal Degeneration; Retinitis Pigmentosa; Rod Outer Segments; Role; Structure; Sucrose; Testing; Tissues; Transgenic Model; Tubular formation; Vertebrate Photoreceptors; Visual; Wild Type Mouse; Work; base; cell type; human PHEMX protein; in vivo; inherited retinal degeneration; insight; interest; mouse development; mouse model; palmitoylation; promoter; radiotracer; research study; retinal rods; sedimentation velocity; transcription factor

DESCRIPTION (provided by applicant): Our research objective is to study the link between Rds (a retinal tetraspanin) and the maintenance of the cone and rod outer segments in order to promote a better understanding of the debilitating retinal degenerations that accompany Rds mutations. In this proposal, I hypothesize that Rds forms an intricate complex of multiple proteins which interact to regulate the outer segment cytoskeleton. The goal of this application is to characterize the differences between Rds complexes in rods vs. cones and examine the way those complexes interact with the cytoskeleton by using cone and rod-dominant mouse models expressing different amounts of Rds. Two aims are proposed: in Aim 1 I will determine what differences exist in the protein composition of Rds complexes in rods and cones. I will use radiolabeling and affinity purification to identify Rds interacting proteins and palmitoylation status in rods (using the rod only Crx-Nrl mouse) and cones (using the Nrl"'" mouse). Further, I will employ biochemical and structural methods such as reciprocal co-IP, velocity sedimentation, and immunohistochemistry/confocal microscopy to characterize Rds binding partners identified by preliminary proteomic studies on affinity purified Rds complexes from bovine outer segments. The goal of Aim 2 is to determine whether Rds complexes participate in regulation of the outer segment cytoskeleton. I hypothesize that Rds is needed for the proper formation of the outer segment rim microdomain and that this microdomain is necessary for the proper localization of proteins that regulate the cytoskeleton and adhesion complexes. This aim will include biochemical and structural experiments using several unique mouse models which express varying amounts of Rds in rods (Crx-Nrl, Crx-Nrl/rc/s+A, Crx-Nrl/rctev') and cones (Nrl"'', Nrl^/rcfe* Nrf/L/rcfe-/). In addition to the core scientific aims of this project, an additional purpose of this work is to give me an experimental framework within which I can develop into an independent researcher. In keeping with the goals of the National Eye Institute, this application is directly relevant to public health. Rds mutations can cause severe visual defects and there are no curative treatments for inherited retinal degenerations currently available. The project will provide important insight into the cell biology of outer segment formation particularly regarding the role of Rds in that process. Further understanding of outer segment morphogenesis, particularly in the less well-studied cone, is critical both for understanding the disease process in the human retina and for developing new treatments for macular degenerations.

描述(申请人提供)：我们的研究目标是研究RDS(一种视网膜四肽)与视锥和视杆外段的维持之间的联系，以促进更好地了解伴随RDS突变而导致的视网膜衰弱退化。在这个提议中，我假设RDS形成了一个由多种蛋白质组成的复杂复合体，这些蛋白质相互作用来调节外层细胞骨架。这项应用的目的是通过使用表达不同数量RD的视锥和视锥优势小鼠模型来表征视杆和视锥中RDS复合体之间的差异，并检查这些复合体与细胞骨架相互作用的方式。提出了两个目标：在目标1中，我将确定视杆和视锥中RDS复合体的蛋白质组成存在哪些差异。我将使用放射性标记和亲和纯化来鉴定RDS相互作用蛋白和杆状(仅使用杆状CRX-NRL小鼠)和视锥(使用NRL‘’小鼠)中的棕榈酰化状态。此外，我将使用生化和结构方法，如互易共IP、速度沉淀和免疫组织化学/共聚焦显微镜来鉴定通过初步蛋白质组学研究鉴定的牛外节Rds亲和纯化Rds复合体的Rds结合伙伴。目标2的目的是确定RDS复合体是否参与外段细胞骨架的调节。我假设RDS是正确形成外段RIM微域所必需的，并且这个微域对于调节细胞骨架和黏附复合体的蛋白质的正确定位是必要的。这一目标将包括使用几种独特的小鼠模型进行生化和结构实验，这些模型在视杆细胞(CRX-NRL，CRX-NRL/RC/S+A，CRX-NRL/Rctev‘)和视锥(NRL“’，NRL^/RCfE*Nrf/L/RCfE-/)中表达不同数量的RD。除了这个项目的核心科学目标之外，这项工作的另一个目的是给我一个实验框架，在这个框架内我可以发展成为一名独立的研究人员。为了与国家眼科研究所的目标保持一致，这项应用与公共卫生直接相关。RDS突变会导致严重的视觉缺陷，目前还没有治疗遗传性视网膜变性的有效方法。该项目将为外段形成的细胞生物学提供重要的见解，特别是关于RD在这一过程中的作用。进一步了解外节的形态发生，特别是在研究较少的视锥细胞中，对于了解人类视网膜的疾病过程和开发治疗黄斑变性的新方法都是至关重要的。