Fiber Cell Cytoskeletal Scaffolds-Role in Lens Architecture and Function

纤维细胞细胞骨架支架-在晶状体结构和功能中的作用

• 批准号: 8184590
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2013-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8184590
• 关键词: Actins; Adhesions; Adhesives; Affect; Animal Model; Ankyrins; Antibodies; Architecture; Biochemical; Cataract; Cell Communication; Cell Differentiation process; Cell Maturation; Cell Shape; Cell membrane; Cells; Cloning; Complex; Connexins; Crystalline Lens; Data; Differentiation and Growth; Disease; Exhibits; Goals; Growth and Development function; Immunoprecipitation; Intercellular Junctions; Knockout Mice; Label; Lens Fiber; MIP gene; Maintenance; Mass Spectrum Analysis; Mechanics; Mediating; Membrane; Modeling; Molecular; Mus; Myelin Sheath; N-terminal; Optical Coherence Tomography; Optics; Phenotype; Play; Property; Protein Isoforms; Proteins; Research; Research Project Grants; Resolution; Role; Scaffolding Protein; Scanning Transmission Electron Microscopy Procedures; Schwann Cells; Screening procedure; Shapes; Spectrin; Techniques; Tertiary Protein Structure; Testing; Three-Dimensional Imaging; Work; Yeasts; ezrin; fiber cell; filensin; immunoaffinity chromatography; innovation; insight; lens; lens transparency; macromolecule; moesin; mouse model; novel; periaxin; radixin protein; rho GTP-Binding Proteins; scaffold; yeast two hybrid system; Actins; Adhesions; Adhesives; Affect; Animal Model; Ankyrins; Antibodies; Architecture; Biochemical; Cataract; Cell Communication; Cell Differentiation process; Cell Maturation; Cell Shape; Cell membrane; Cells; Cloning; Complex; Connexins; Crystalline Lens; Data; Differentiation and Growth; Disease; Exhibits; Goals; Growth and Development function; Immunoprecipitation; Intercellular Junctions; Knockout Mice; Label; Lens Fiber; MIP gene; Maintenance; Mass Spectrum Analysis; Mechanics; Mediating; Membrane; Modeling; Molecular; Mus; Myelin Sheath; N-terminal; Optical Coherence Tomography; Optics; Phenotype; Play; Property; Protein Isoforms; Proteins; Research; Research Project Grants; Resolution; Role; Scaffolding Protein; Scanning Transmission Electron Microscopy Procedures; Schwann Cells; Screening procedure; Shapes; Spectrin; Techniques; Tertiary Protein Structure; Testing; Three-Dimensional Imaging; Work; Yeasts; ezrin; fiber cell; filensin; immunoaffinity chromatography; innovation; insight; lens; lens transparency; macromolecule; moesin; mouse model; novel; periaxin; radixin protein; rho GTP-Binding Proteins; scaffold; yeast two hybrid system

DESCRIPTION (provided by applicant): The transparency and normal shape of the ocular lens are dependent upon the precise hexagonal geometry, ordered packing, deformability, and membrane organization of highly elongated fiber cells. The broad goals of this research project are to identify and characterize the membrane cytoskeletal scaffolding proteins regulating lens fiber cell shape, cell-cell interactions, membrane stability and organization, and to determine how dysregulation of this scaffolding activity impacts lens transparency and function in animal models. This proposal will investigate the function(s) of the PDZ domain protein-Periaxin, a component of the major cortical adhesive complex-EPPD (ezrin-periaxin-periplakin-desmoyokin) in lens fiber cells. Recent work on characterization of the EPPD complex in the mouse lens led to our discovery that lens fibers exhibit preferential expression of periaxin (PRX), a protein originally thought to be expressed only in myelinating Schwann cells, where it mediates membrane stabilization. PRX expression is robustly induced during fiber cell differentiation, and the protein clusters discretely at the tri-cellular junctions in mature lens fibers. Interestingly, co-immunoprecipitates of PRX from lens fibers contain ankyrin B, spectrin, filensin, desmoyokin, NrCAM and aquaporin-0, suggesting a role for PRX in fiber cell membrane network connectivity, adhesive interactions, and membrane subdomain organization. Significantly, preliminary data from PRX null mice revealed disruptions of fiber cell shape, packing, and membrane integrity but no effects on lens development and growth, arguing a vital role for PRX in maintenance of lens phenotype and function. These significant and novel observations prompted us to hypothesize that the scaffolding interactions of PRX play a key role in stabilization of lens fiber cell tri-cellular junctions, hexagonal geometries, and membrane integrity which are crucial for maintenance of lens transparency and function. For a mechanistic understanding of the role of PRX in mediating the above mentioned functions, three specific aims will be investigated in this application: 1. Analysis of the distribution of different PRX isoforms with unique N-terminal sequences in mouse lens fibers during maturation and compaction, using high resolution confocal 3D imaging; 2. Characterization of PRX scaffolding activity in stabilization of fiber cell tri-cellular junctions and membrane organization via identification of PRX interacting proteins using immunoprecipitation, mass spectrometry and yeast two-hybrid analysis; 3. Determination of how absence of PRX affects lens optical quality, fiber cell shape, packing and membrane integrity using PRX null and conditional knockout mouse lenses. The completion of these studies is expected to provide novel insights into the broad significance of cortical cytoskeletal scaffolding activity and the specific role of periaxin in maintaining lens fiber cell shape, packing, deformability, cell-cell junctions, and membrane integrity in both normal and cataractous lenses. PUBLIC HEALTH RELEVANCE: Transparency and normal shape are critical for ocular lens function, and rely on the precise packing, deformability, and membrane integrity of hexagonal fiber cells. This application will examine the importance of the cytoskeletal scaffolding activity of periaxin in maintaining lens fiber cell shape, arrangement, and membrane organization using mouse lenses that lack this protein. This project is expected to provide significant insights into the cytoskeletal scaffolding mechanisms regulating lens fiber cell shape, adhesive interactions, arrangement, and membrane organization, and how they may impact optical clarity and function of normal and cataractous lenses.

描述（由申请人提供）：眼镜的透明度和正常形状取决于高度伸长的纤维细胞的精确六边形几何形状，有序填充，变形性和膜组织。该研究项目的广泛目标是识别和表征膜细胞骨架脚手架蛋白质，调节晶状体纤维细胞形状，细胞 - 细胞相互作用，膜稳定性和组织，并确定这种脚手架活性的失调如何影响动物模型中的透镜透明度和功能。该建议将研究晶状体纤维细胞中主要皮质粘合剂复合物eppd（Ezrin-透露蛋白蛋白 - 周期性 - 透露蛋白酶）的PDZ结构蛋白蛋白质周期素的功能。 关于EPPD复合物在小鼠透镜中的表征的最新研究导致我们发现透镜纤维表现出纤维纤维的优先表达（PRX），这种蛋白最初被认为仅在髓鞘的schwann细胞中表达，从而介导膜稳定性。在纤维细胞分化过程中，PRX表达可牢固诱导，并且在成熟晶状体纤维的三个细胞连接处离散地蛋白簇。有趣的是，来自镜头纤维的PRX的共免疫沉淀物包含Ankyrin B，Spectrin，Filensin，Desmoyokin，NrCAM和Aquaporin-0，这表明PRX在纤维细胞膜网络连接性，粘合剂相互作用，粘合剂相互作用以及膜子域中的作用。值得注意的是，来自PRX无效小鼠的初步数据显示，纤维细胞形状，填料和膜完整性的中断，但对晶状体发育和生长没有影响，认为PRX在维持晶状体表型和功能方面至关重要。这些重要而新颖的观察结果促使我们假设PRX的脚手架相互作用在稳定晶状体纤维细胞三个细胞连接，六边形几何形状和膜完整性方面起着关键作用，这对于维持透镜透明度和功能至关重要。 为了理解PRX在介导上述功能中的作用的机械理解，将在此应用中研究三个具体目标：1。分析在成熟和压缩过程中，在成熟和压缩过程中，具有独特的N末端序列的不同PRX同工型的分布，使用高分辨率共溶液共溶液共线3D成像； 2。通过使用免疫沉淀，质谱法和酵母两杂交分析来鉴定PRX相互作用蛋白，在纤维细胞三细胞连接和膜组织稳定中表征PRX脚手架活性。 3。确定PRX的缺失如何使用PRX NULL和有条件的基因敲除小鼠透镜影响镜头光学质量，纤维细胞形状，填充和膜完整性。 预计这些研究的完成将提供对皮质细胞骨骼支架活性的广泛意义的新见解，以及周围素在维持晶状体纤维细胞形状，堆积，可变形性，细胞 - 细胞连接和膜完整性中在正常和心电图中的特定作用。 公共卫生相关性：透明度和正常形状对于眼镜晶状体功能至关重要，并且依赖于六边形纤维细胞的精确填料，变形性和膜完整性。该应用将使用缺乏该蛋白质的小鼠透镜来研究周围素的细胞骨架脚手架活性在维持晶状体纤维细胞形状，排列和膜组织中的重要性。预计该项目将提供有关调节透镜纤维细胞形状，粘合剂相互作用，布置和膜组织的细胞骨架脚手架机制的重要见解，以及它们如何影响正常和白内障透镜的光学清晰度和功能。