Cell Junctions and Cell Membranes in the Lens

描述（由申请人提供）：本项目的长期目标是阐明脂筏结构域在透镜纤维细胞膜及其相关蛋白中的功能作用，这些蛋白对维持透镜的透明度至关重要。在许多类型的细胞中，脂筏与各种膜过程如突起、微绒毛、丝状伪足和板状伪足功能相关。脂筏作为平台募集蛋白质进行信号转导、钙转运和胆固醇转运。在所有种类的透镜纤维细胞中，大量的联锁域（以球窝、突起和微绒毛突起的形式）突出到相邻的细胞中并增加细胞膜表面积。由于透镜是一种血管器官，这种独特的互锁结构域（ID）的结构配置将是促进细胞之间分子交换的理想选择。本课题主要研究鸡胚胎和成年晶状体内脂质筏的关键脂质和蛋白质成分。由于ID与其他细胞的脂筏具有一些独特的特征，我们假设晶状体特异性ID是透镜纤维细胞中的脂筏。目的1测试从这个假设的预测，胆固醇是丰富的各种ID，通过使用菲律宾细胞化学与冷冻断裂TEM。目的2：利用免疫金电镜结合高压冷冻技术，验证小窝蛋白1、PMCA、Src激酶等关键性筏相关蛋白在诱导分化过程中的富集预测。目的3：通过冷冻断裂免疫金复制标记（FRIL）和生物化学方法，验证我们假设的推论，即球窝结构域和扁平细胞膜中的cav-1-连接蛋白相互作用对透镜纤维中间隙连接的组装和成熟至关重要。具体而言，cav-1在胆固醇运输中的作用将被检查，以在连接组装、成熟和老化期间将富含胆固醇的间隙连接转化为不含胆固醇的间隙连接，使用我们的创新方法，在EM水平将菲律宾细胞化学与FRIL相结合。将应用定量分析来评估cav-1与含胆固醇的间隙连接的关联，以确定它们作为透镜中的脂筏结构域的假定作用。由于脂筏的功能作用严重依赖于它们的细胞骨架组织，因此Aim 4将确定肌动蛋白分支网络构型并鉴定肌动蛋白相关蛋白（例如，Arp 2/3，ezrin和Eschemmin）在发展筏状结构域，通过使用高压冷冻程序。

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the functional roles of lipid raft domains in lens fiber-cell membranes, and their associated proteins, which are crucial in maintaining the transparency of the lens. In many types of cells, lipid rafts are functionally associated with various membrane processes such as protrusions, microvilli, filopodia and lamellipodia. The lipid rafts function as platforms to recruit proteins for signal transduction, calcium transport and cholesterol transport. In lens fiber cells of all species, large numbers of interlocking domains (in the forms of ball-and-socket, protrusion and microvillus processes) protrude into neighboring cells and increase cell membrane surface area. Since the lens is a vascular organ, this unique structural configuration of interlocking domains (IDs) would be ideal for facilitating molecular exchange between cells. In this project, we focus on identification of key lipid and protein components of lipid rafts in IDs of embryonic and adult chicken lenses. Because IDs share several unique features with lipid rafts of other cells, we hypothesize that the lens-specific IDs are the lipid rafts in lens fiber cells. Aim 1 tests the prediction from this hypothesis that cholesterol is enriched in various IDs, by using filipin cytochemistry with freeze-fracture TEM. Aim 2 tests the prediction that caveolin-1, PMCA, Src kinase and other key raft-associated proteins are enriched in IDs, by using immunogold EM with high-pressure freezing procedures. Aim 3 tests the corollary of our hypothesis that cav-1-connexin interaction in ball-and- socket domains and flat cell membranes is critical for gap junction assembly and maturation in lens fibers, by using freeze-fracture immunogold replica labeling (FRIL) and biochemistry. Specifically, the roles of cav-1 in trafficking of cholesterol will be examined for the transformation of cholesterol-rich to cholesterol-free gap junctions during junction assembly, maturation, and aging using our innovative methodology combining filipin cytochemistry with FRIL at EM level. Quantitative analysis will be applied for assessing the association of cav-1 with cholesterol- containing gap junctions for their putative role as lipid-raft domains in the lens. Because the functional roles of lipid rafts critically depend on their cytoskeletal organization, Aim 4 will determine the actin branching network configuration and identify the actin-associated proteins (e.g., Arp2/3, ezrin and paralemmin) in developing raft-like domains, by using high pressure freezing procedures.