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

基本信息

批准号：
7752541
负责人：
Shannon M Conley
金额：
$ 5.38万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
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/7752541
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 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

项目摘要

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（视网膜四翼烷蛋白）与锥形和杆外部段之间的联系，以便更好地理解RDS突变伴随RDS突变的视网膜变性。在此提案中，我假设RDS形成了多种蛋白质的复杂复合物，该复合物相互作用以调节外部段细胞骨架。该应用的目的是表征杆与锥体中RDS复合物之间的差异，并通过使用锥体和杆位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，从而表达不同量的RDS。提出了两个目的：在AIM 1中，我将确定杆和锥体中RDS复合物蛋白质组成中存在哪些差异。我将使用放射性标记和亲和力纯化来识别RDS相互作用的蛋白质和棕榈酰化状态（使用仅杆CRX-NRL小鼠）和锥（使用NRL“'”小鼠）。此外，我将采用生化和结构方法，例如相互co-IP，速度沉积和免疫组织化学/共聚焦显微镜，以表征通过蛋白质组学研究对亲和力纯化纯化纯化的RDS纯化纯化RDS复合物的RDS结合伴侣的表征。目标2的目的是确定RDS复合物是否参与了外部段细胞骨架的调节。我假设需要RDS才能正确形成外部段边缘微域，并且该微域对于正确定位调节细胞骨架和粘附复合物的蛋白质是必需的。此目标将包括使用几种独特的鼠标模型的生化和结构实验，这些鼠标模型表达了不同量的RD（CRX-NRL，CRX-NRL/RC/RC/S+A，CRX-NRL/RCTEV'）和CONES（NRL“''''，NRL^/RCFE* NRF* NRF/RCFE）。此外，此外还有一项核心。为我提供一个实验框架，我可以将其发展成一个独立的研究人员。经过良好的锥体，对于理解人类视网膜的疾病过程和开发新的黄斑变性疗法至关重要。