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: 8975207
• 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/8975207
• 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; Health; Image; Immunoprecipitation; 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; 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; mouse model; mutant; novel; null mutation; periaxin; ponsin; protein function; protein protein interaction; 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(申请人提供)：透明度和调节能力是人工晶状体聚焦图像的关键决定因素，取决于纤维细胞的精确六角几何形状、径向堆积、机械性能和膜组织。本研究项目的主要目标是鉴定和鉴定调控纤维细胞形态、黏附、拉伸特性和膜组织的质膜细胞骨架蛋白，并确定膜细胞骨架活性的失调如何影响晶状体的这些重要特性。本研究将从晶状体功能的角度研究环磷酰胺(Periaxin，Prx)及其相互作用蛋白Ankyrin-B(AnkB)和抗肌营养不良蛋白-糖蛋白复合体(DGc)在分化和成熟纤维细胞中的协调和相互依赖的支架功能(S)。我们最近的观察表明，Prx和AnkB不仅以相互的方式进行免疫共沉淀，而且在纤维细胞中的分布特征也显示出密切的空间重叠。重要的是，携带Prx缺失或杂合子的小鼠 AnkB零突变(AnkB-/+)在连接蛋白-50、纤丝蛋白、血影蛋白、NrCAM和WAVE-2的纤维细胞形态、排列和膜聚集方面表现出显著和相似的破坏。此外，在AnkB-/+晶状体中，Prx不能与质膜结合，反之，在Prx-/+晶状体中，AnkB水平降低。在这些晶状体中，DGC的膜组织明显受损，表明PRX、AnkB和DGC之间存在密切的相互作用。尽管Prx Null和AnkB-/+小鼠晶状体基本保持透明，但晶状体硬度显著降低。这些多重而重要的初步观察促使我们假设，纤维特异的PDZ结构域蛋白Prx通过与AnkB和DGC直接相互作用，作为关键的支架蛋白，维持纤维细胞的六角形形状、径向和紧凑的排列、膜域组织和机械性能，这些都是晶状体透明度和调节的关键决定因素。为了确定Prx-AnkB-DGC轴在晶状体组织中协调和必要的支架作用(S)的功能重要性，我们将追求以下三个具体目标：1.详细描述Prx、AnkB和DGC在小鼠晶状体纤维中相互依赖的支架活性；2.利用基因靶向小鼠在体内详细分析Prx、AnkB的缺失和DGC活性降低对晶状体光学质量、纤维细胞形状、黏附和膜骨架网络的影响；3.确定Prx-AnkB-DGC轴在组织和调节晶状体纤维细胞通道蛋白膜结构域和活性中的作用(S)，并使用基因靶向的小鼠来维持晶状体的机械性能。这些未被研究的纤维细胞方面的完成有望为膜细胞骨架相互作用如何调节人工晶状体的光学特性和调节能力提供新的和重要的见解。