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）与视锥和视杆外节维持之间的联系，以促进更好地理解伴随Rds突变的衰弱性视网膜变性。在这个建议中，我假设Rds形成了一个复杂的复杂的多个蛋白质相互作用，以调节外节细胞骨架。本申请的目的是表征视杆细胞与视锥细胞中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占优势的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，我将确定在杆和锥中的Rds复合物的蛋白质组成存在什么差异。我将使用放射性标记和亲和纯化来鉴定视杆细胞（仅使用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的方法，如互惠共IP，速度沉降，和免疫组织化学/共聚焦显微镜表征Rds结合伴侣的初步蛋白质组学研究亲和纯化的Rds复合物从牛的外段。目的2的目标是确定Rds复合物是否参与调节外节细胞骨架。我假设，Rds是需要适当形成的外段边缘微域，这个微域是必要的适当定位的蛋白质，调节细胞骨架和粘附复合物。该目的将包括使用几种独特的小鼠模型的生物化学和结构实验，所述小鼠模型在视杆（Crx-Nrl，Crx-Nrl/rc/s+A，Crx-Nrl/rctev '）和视锥（Nrl "/“，Nrl/rcfe* Nrf/L/rcfe-/）中表达不同量的Rds。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突变可导致严重的视力缺陷，目前还没有治疗遗传性视网膜变性的有效方法。该项目将提供重要的洞察外节形成的细胞生物学，特别是关于Rds在该过程中的作用。进一步了解外节形态发生，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黄斑变性的新治疗方法都至关重要。