Cell Junctions and Cell Membranes in the Lens

描述(申请人提供)：该项目的长期目标是阐明晶状体纤维细胞膜中脂筏结构域及其相关蛋白质的功能作用，这对维持晶状体的透明度至关重要。在许多类型的细胞中，脂筏在功能上与各种膜过程有关，如突起、微绒毛、丝状伪足和片状伪足。脂筏的功能是为信号转导、钙转运和胆固醇转运招募蛋白质的平台。在所有种类的晶状体纤维细胞中，大量的互锁结构域(以球窝、突起和微绒毛突起的形式)突出到相邻的细胞中，并增加细胞膜的表面积。由于晶状体是一个血管器官，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。在本项目中，我们着重于鉴定胚胎和成年鸡晶状体中脂筏的关键脂类和蛋白质成分。由于ID与其他细胞的脂筏有几个共同的独特特征，我们假设晶状体特异性ID是晶状体纤维细胞中的脂筏。目的1利用冷冻断裂的细胞化学技术，从这一假说中验证胆固醇在不同的IDs中富含的预测。目的2利用免疫金EM结合高压冷冻程序，验证小窝蛋白-1、PMCA、Src蛋白和其他关键的RAFT相关蛋白富含IDs的预测。目的3用冷冻断裂免疫金标记法和生化方法验证我们的假设的推论，即球窝结构域和扁平细胞膜上的Cav-1-连接蛋白相互作用对晶状体纤维的缝隙连接组装和成熟至关重要。具体地说，我们将使用我们在EM水平上结合Filipin细胞化学和FRIL的创新方法，研究Cav-1在胆固醇运输中的作用，以期在连接组装、成熟和老化过程中将高胆固醇缝隙连接转化为无胆固醇缝隙连接。定量分析将用于评估Cav-1与含胆固醇的缝隙连接之间的关联，因为它们可能在晶状体中扮演脂筏结构域的角色。由于脂筏的功能在很大程度上依赖于它们的细胞骨架组织，因此Aim 4将通过高压冷冻程序确定肌动蛋白分支网络的结构，并确定肌动蛋白相关蛋白(如Arp2/3、Ezrin和Paralemmin)在形成RAFT样结构域中的作用。

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.