Fiber Cell Membrane Organization-Role in Lens Architecture and Function

纤维细胞膜组织-在晶状体结构和功能中的作用

• 批准号: 9180703
• 负责人: P VASANTHA Rao
• 金额: $ 39.25万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9180703
• 关键词: ANK2 gene; Actins; Address; Adhesions; Adhesives; Affect; Architecture; Area; Biochemical; Biological Assay; Blood Circulation; Calcium; Cataract; Cell Communication; Cell Shape; Cell membrane; Cell-Cell Adhesion; Cells; Characteristics; Complex; Connexins; Coupling; Crystalline Lens; Cytoskeletal Modeling; Cytoskeleton; Dystroglycan; Dystrophin; Etiology; Exhibits; Fiber; Gene Targeting; Genes; Geometry; Glycoproteins; Goals; Image; Immunoprecipitation; Impairment; In Vitro; Knock-in Mouse; Knock-out; Knockout Mice; L-Type Calcium Channels; Lens Fiber; MIP gene; Maintenance; Mechanics; Membrane; Membrane Proteins; Molecular; Mus; Myelin Sheath; Ocular Fixation; Optical Coherence Tomography; Optics; Phosphorylation; Physiological; Play; Property; Proteins; Proteomics; Radial; Regulation; Research; Research Project Grants; Resolution; Role; Scaffolding Protein; Scanning; Shapes; Spectrin; Structure; Tertiary Protein Structure; Testing; Tissues; Transmission Electron Microscopy; Visual Accommodation; Work; age related; base; fiber cell; filensin; in vivo; innovation; insight; lens; lens intrinsic protein MP 70; lens transparency; mechanical properties; mouse model; mutant; novel; null mutation; periaxin; ponsin; protein B; protein function; protein protein interaction; public health relevance; reconstruction; scaffold; skeletal

 DESCRIPTION (provided by applicant): Transparency and accommodation are crucial determinants of image focusing by the ocular lens and depend upon precise hexagonal geometry, radial packing, mechanical properties and membrane organization of fiber cells. The broad goals of this research project are to identify and characterize plasma membrane cytoskeletal scaffolding proteins regulating fiber cell shape, adhesion, tensile properties, membrane organization, and to determine how dysregulation of membrane cytoskeletal scaffolding activity impacts these vital characteristics of lens. This proposal will investigate th coordinated and interdependent scaffolding function(s) of Periaxin (PRX) and its interacting proteins- Ankyrin-B (AnkB) and the dystrophin- glycoprotein complex (DGC) in differentiating and mature fiber cells in the context of lens function. Our recent observations show that PRX and AnkB not only co-immunoprecipitate in a reciprocal manner but also exhibit a close spatial overlap of distribution profile in fiber cells. Importantly, mice with PRX null or heterozygous for AnkB null mutations (AnkB-/+) revealed prominent and similar disruptions in fiber cell shape, alignment and membrane clustering of connexin-50, filensin, spectrin, NrCAM and WAVE-2. Moreover, in AnkB-/+ lenses, PRX failed to anchor to the plasma membrane and in a vice versa manner, AnkB levels were decreased in PRX null lenses. Significantly in these lenses, membrane organization of the DGC was dramatically impaired indicating a close interaction among PRX, AnkB and the DGC. Although both PRX null and AnkB-/+ mouse lenses remained largely transparent, lens stiffness was found to be significantly decreased. These multiple and significant preliminary observations prompted us to hypothesize that the fiber specific PDZ- domain protein PRX serves as a key scaffolding protein via direct interaction with AnkB and the DGC, to maintain fiber cell hexagonal shape, radial and compact alignment, membrane domain organization and mechanical properties, which are critical determinants of lens transparency and accommodation. To determine the functional importance of the coordinated and essential scaffolding role(s) played by the PRX-AnkB-DGC axis in lens tissue, we will pursue the following three specific aims: 1. Detailed in vitro characterization of the interdependent scaffolding activities of PRX, AnkB and the DGC in mouse lens fibers, 2. Detailed in vivo analysis of how absence of PRX, AnkB and impaired DGC activity affects lens optical quality, fiber cell shape, adhesion and the membrane skeletal network, using gene targeted mice, and 3. Determine the role(s) of PRX-AnkB-DGC axis in organization and regulation of lens fiber cell channel protein membrane domains and activity, and in maintenance of lens mechanical properties using gene targeted mice. The completion of these unstudied aspects of fiber cells is expected to provide novel and important insights into how membrane cytoskeletal scaffolding interactions regulate the unique structure-function attributes of ocular lens in the context of its optical properties an accommodation.

 描述（由申请人提供）：透明度和适应性是通过眼透镜聚焦图像的关键决定因素，并取决于精确的六边形几何形状、径向填充、机械性能和纤维细胞的膜组织。本研究项目的主要目标是鉴定和表征调节纤维细胞形状、粘附性、拉伸性质、膜组织的质膜细胞骨架支架蛋白，并确定膜细胞骨架支架活性失调如何影响透镜的这些重要特征。本研究将探讨在透镜功能的背景下，Periaxin（PRX）及其相互作用蛋白-AnkB（AnkB）和Dystrophin-糖蛋白复合物（DGC）在纤维细胞分化和成熟中的协调和相互依赖的支架功能。 我们最近的观察表明，PRX和AnkB不仅以相互的方式共免疫沉淀，而且在纤维细胞中的分布曲线也表现出密切的空间重叠。重要的是，PRX缺失或杂合的小鼠 AnkB无效突变（AnkB-/+）揭示了纤维细胞形状、连接蛋白-50、filensin、血影蛋白、NrCAM和WAVE-2的排列和膜聚集的显著和相似的破坏。此外，在AnkB-/+镜片中，PRX未能锚于质膜，反之亦然，在PRX无效镜片中AnkB水平降低。在这些晶状体中，DGC的膜组织显著受损，表明PRX、AnkB和DGC之间存在密切的相互作用。尽管PRX无效和AnkB-/+小鼠晶状体均保持大部分透明，但发现透镜刚度显著降低。这些多个和重要的初步观察结果促使我们假设纤维特异性PDZ结构域蛋白PRX通过与AnkB和DGC直接相互作用充当关键支架蛋白，以维持纤维细胞六边形形状、径向和紧凑排列、膜结构域组织和机械性质，这些是透镜透明度和调节的关键决定因素。 为了确定由PRX-AnkB-DGC轴在透镜组织中发挥的协调和必要的支架作用的功能重要性，我们将追求以下三个具体目标：PRX、AnkB和DGC在小鼠透镜纤维中的相互依赖的支架活性的详细体外表征，2.使用基因靶向小鼠，PRX、AnkB和受损的DGC活性的缺乏如何影响透镜光学质量、纤维细胞形状、粘附和膜骨架网络的详细体内分析，和3.使用基因靶向小鼠确定PRX-AnkB-DGC轴在透镜纤维细胞通道蛋白膜结构域和活性的组织和调节中以及在透镜机械性质的维持中的作用。 完成这些未研究的纤维细胞方面，预计将提供新的和重要的见解膜细胞骨架相互作用如何调节独特的结构-功能属性的眼透镜的上下文中，其光学特性的调节。