Fiber Cell Cytoskeletal Scaffolds-Role in Lens Architecture and Function

描述（由申请人提供）：晶状体的透明度和正常形状取决于高度拉长的纤维细胞精确的六边形几何形状、有序的包装、可变形性和膜组织。本研究项目的主要目标是鉴定和表征调节晶状体纤维细胞形状、细胞间相互作用、膜稳定性和组织的膜细胞骨架支架蛋白，并确定这种支架活性的失调如何影响动物模型中晶状体的透明度和功能。本研究将研究晶状体纤维细胞中主要皮质黏附复合物eppd （ezrin-periaxin-periplakin-desmoyokin）的组成部分PDZ结构域蛋白-periaxin的功能。最近对小鼠晶状体中EPPD复合物的表征工作使我们发现晶状体纤维表现出周轴蛋白（PRX）的优先表达，这种蛋白最初被认为只在髓鞘雪旺细胞中表达，在那里它介导膜稳定。PRX的表达在纤维细胞分化过程中被强烈诱导，并且在成熟晶状体纤维的三细胞连接处离散地聚集。有趣的是，来自晶态纤维的PRX共免疫沉淀含有锚蛋白B、谱蛋白、纤维素、粘蛋白、NrCAM和水通道蛋白0，这表明PRX在纤维细胞膜网络连接、粘附相互作用和膜亚域组织中发挥作用。值得注意的是，来自PRX小鼠的初步数据显示，纤维细胞形状、包装和膜完整性受到破坏，但对晶状体发育和生长没有影响，这表明PRX在维持晶状体表型和功能方面起着至关重要的作用。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晶状体纤维细胞三细胞连接、六边形几何形状和膜完整性的稳定中起关键作用，这对维持晶状体的透明度和功能至关重要。为了从机制上理解PRX在调节上述功能中的作用，本应用将研究三个具体目标：1。利用高分辨率共聚焦三维成像技术分析小鼠晶状体纤维成熟和压实过程中具有独特n端序列的不同PRX亚型的分布；2. 通过免疫沉淀、质谱和酵母双杂交分析鉴定PRX相互作用蛋白，表征PRX支架在纤维细胞三细胞连接和膜组织稳定中的活性；3. 使用PRX无效和条件敲除小鼠透镜确定PRX缺失如何影响透镜光学质量、纤维细胞形状、包装和膜完整性。这些研究的完成有望为皮层细胞骨架支架活性的广泛意义以及周轴蛋白在维持正常和白内障晶状体中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方面的特定作用提供新的见解。

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.