Fiber Cell Cytoskeletal Scaffolds-Role in Lens Architecture and Function

描述（由申请人提供）：眼透镜的透明度和正常形状取决于精确的六边形几何形状、有序堆积、可变形性和高度伸长的纤维细胞的膜组织。该研究项目的主要目标是确定和表征调节透镜纤维细胞形状、细胞-细胞相互作用、膜稳定性和组织的膜细胞骨架蛋白，并确定这种支架活性失调如何影响动物模型中的透镜透明度和功能。本研究将探讨透镜纤维细胞中主要的皮质粘附复合物EPPD（ezrin-periaxin-periplakin-desmoyokin）的一种成分--PDZ结构域蛋白--Periaxin的功能。 最近对小鼠透镜中EPPD复合物的表征工作导致我们发现透镜纤维表现出优先表达的周蛋白（PRX），该蛋白最初被认为仅在髓鞘形成的许旺细胞中表达，在那里它介导膜稳定。PRX表达在纤维细胞分化过程中被强烈诱导，并且在成熟透镜纤维中蛋白质在三细胞连接处离散地聚集。有趣的是，来自透镜纤维的PRX的免疫共沉淀物含有锚蛋白B、血影蛋白、filensin、桥粒蛋白、NrCAM和水通道蛋白-0，这表明PRX在纤维细胞膜网络连接、粘附相互作用和膜子域组织中的作用。值得注意的是，PRX基因敲除小鼠的初步数据显示纤维细胞形状、包装和膜完整性的破坏，但对透镜发育和生长没有影响，这表明PRX在维持透镜表型和功能中起着至关重要的作用。这些重要和新颖的观察结果促使我们假设PRX的支架相互作用在透镜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，这些对于维持透镜的透明度和功能至关重要。 为了从机制上理解PRX在介导上述功能中的作用，本申请将研究三个具体目标：1.使用高分辨率共聚焦3D成像，分析在小鼠透镜纤维成熟和致密过程中具有独特N-末端序列的不同PRX同种型的分布; 2.通过使用免疫沉淀、质谱和酵母双杂交分析鉴定PRX相互作用蛋白来表征PRX在纤维细胞三细胞连接和膜组织稳定中的支架活性; 3.使用PRX无效和条件性敲除小鼠镜片确定PRX缺失如何影响透镜光学质量、纤维细胞形状、填充和膜完整性。 这些研究的完成有望为皮质细胞骨架支架活性的广泛意义和轴周蛋白在维持正常和白内障晶状体中透镜纤维细胞形状、包装、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。 公共卫生相关性：透明性和正常形状对于眼透镜功能是至关重要的，并且依赖于六边形纤维细胞的精确填充、可变形性和膜完整性。本申请将研究的重要性，细胞骨架支架活性的periaxin在保持透镜纤维细胞的形状，安排，和膜组织使用小鼠晶状体，缺乏这种蛋白质。该项目预计将提供重要的见解细胞骨架骨架机制调节透镜纤维细胞的形状，粘附相互作用，安排，和膜组织，以及它们如何可能影响光学清晰度和功能的正常和白内障晶状体。

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.