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（由申请人提供）：我们的研究目标是研究 Rds（一种视网膜四跨膜蛋白）与视锥细胞和视杆细胞外节的维持之间的联系，以促进更好地了解伴随 Rds 突变而导致的视网膜变性。在这个提议中，我假设 Rds 形成了多种蛋白质的复杂复合物，这些蛋白质相互作用来调节外节细胞骨架。本应用的目标是表征视杆细胞和视锥细胞中 Rds 复合物之间的差异，并通过使用表达不同量 Rds 的视锥细胞和视杆细胞主导型小鼠模型来检查这些复合物与细胞骨架相互作用的方式。提出了两个目标：在目标 1 中，我将确定视杆细胞和视锥细胞中 Rds 复合物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识别视杆细胞（仅使用视杆细胞 Crx-Nrl 小鼠）和视锥细胞（使用 Nrl"'" 小鼠）中的 Rds 相互作用蛋白和棕榈酰化状态。此外，我将采用生物化学和结构方法，例如相互共免疫沉淀、速度沉降和免疫组织化学/共聚焦显微镜来表征通过对牛外节亲和纯化的 Rds 复合物进行初步蛋白质组学研究而鉴定的 Rds 结合伴侣。目标 2 的目标是确定 Rds 复合物是否参与外节细胞骨架的调节。我假设 Rds 对于外节边缘微域的正确形成是必需的，并且该微域对于调节细胞骨架和粘附复合物的蛋白质的正确定位是必需的。该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'）和视锥细胞（Nrl"''、Nrl^/rcfe* Nrf/L/rcfe-/）中表达不同数量的 Rd。除了该项目的核心科学目标外，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员。为了与国家眼科研究所的目标保持一致，该应用程序与公共健康直接相关。 Rds 突变可导致严重的视力缺陷，目前尚无针对遗传性视网膜变性的治疗方法。该项目将为外节形成的细胞生物学提供重要的见解，特别是关于 Rds 在此过程中的作用。进一步了解外节形态发生，特别是在较少的情况下 经过充分研究的视锥细胞对于了解人类视网膜的疾病过程和开发黄斑变性的新疗法至关重要。