Fiber Cell Cytoskeletal Scaffolds-Role in Lens Architecture and Function

描述(申请人提供)：人工晶状体的透明度和正常形状取决于高度细长的纤维细胞的精确的六边形几何形状、有序的堆积、变形性和膜组织。该研究项目的主要目标是鉴定和表征调控晶状体纤维细胞形态、细胞-细胞相互作用、膜的稳定性和组织的膜细胞骨架蛋白，并在动物模型中确定这种支架活动的失调如何影响晶状体的透明度和功能。这项建议将研究PDZ结构域蛋白Periaxin的功能(S)，Periaxin是晶状体纤维细胞中主要的皮质黏附复合体EPPD(Ezrin-Periaxin-Periplakin-desmoyokin)的组成部分。最近对小鼠晶状体中EPPD复合体的表征工作导致我们发现晶状体纤维优先表达周轴素(Prx)，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。在纤维细胞分化过程中，Prx的表达被强烈地诱导，在成熟的晶状体纤维中，Prx蛋白分散地聚集在三个细胞的连接处。有趣的是，来自晶状体纤维的Prx免疫共沉淀物含有Ankyrin B、Spectrin、Filensin、Desmoyokin、NrCAM和Aquaporin-0，这表明Prx在纤维细胞膜网络连通性、粘附性相互作用和膜亚结构域组织中发挥作用。值得注意的是，来自Prx基因缺失小鼠的初步数据显示，纤维细胞的形状、堆积和膜完整性受到破坏，但对晶状体的发育和生长没有影响，这表明Prx在维持晶状体的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的支架相互作用在稳定晶状体纤维细胞三细胞连接、六角形几何结构和膜完整性方面发挥了关键作用，而这些对维持晶状体的透明度和功能至关重要。为了从机制上了解Prx在调节上述功能中的作用，我们将在这项应用中研究三个具体目标：1.使用高分辨率共聚焦3D成像，分析不同N端序列的Prx异构体在小鼠晶状体纤维成熟和致密过程中的分布；2.通过免疫沉淀、质谱分析和酵母双杂交分析鉴定Prx相互作用蛋白，表征Prx在稳定纤维细胞三细胞连接和膜组织中的活性；3.使用Prx零和条件基因敲除小鼠晶状体，确定缺少Prx对晶状体光学质量、纤维细胞形状、包装和膜完整性的影响。这些研究的完成有望为皮质细胞骨架活性的广泛意义以及周轴素在维持正常晶状体和白内障晶状体的纤维细胞形状、堆积、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。 与公众健康相关：透明和正常的形状对眼晶状体的功能至关重要，依赖于六角形纤维细胞的精确填充、可变形性和膜的完整性。这项应用将使用缺乏这种蛋白质的小鼠晶状体，研究周轴素的细胞骨架活性在维持晶状体纤维细胞形状、排列和膜组织方面的重要性。该项目有望为调节晶状体纤维细胞形状、粘附剂相互作用、排列和膜组织的细胞骨架机制以及它们如何影响正常和白内障晶状体的光学清晰度和功能提供重要的见解。

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.