Cell Junctions and Cell Membranes in the Lens

晶状体中的细胞连接和细胞膜

• 批准号: 8126324
• 负责人: WOO-KUEN K LO
• 金额: $ 26.61万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8126324
• 关键词: Actins; Adult; Aging; Area; Biochemistry; Blood Vessels; Calcium; Cataract; Cell membrane; Cells; Chickens; Cholesterol; Complex; Connexins; Connexon; Cytoplasm; Cytoskeletal Modeling; Embryo; Fiber; Figs - dietary; Filipin; Filopodia; Fracture; Freeze Fracturing; Freezing; Frequencies; Gap Junctions; Goals; Homeostasis; Intercellular Junctions; Knowledge; Label; Lens Fiber; Lipids; Maintenance; Membrane; Membrane Lipids; Membrane Microdomains; Membrane Proteins; Methodology; Molecular; Organ; Participant; Play; Population; Procedures; Process; Proteins; Recruitment Activity; Research Personnel; Role; Signal Transduction; Site; Sphingomyelins; Surface; Techniques; Testing; Tissues; caveolin 1; cell type; cellular microvillus; cholesterol trafficking; cholesterol-binding protein; cytochemistry; ezrin; fiber cell; immunocytochemistry; innovation; lens; lens transparency; paralemmin; particle; pressure; programs; scaffold; src-Family Kinases

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.

描述（由申请人提供）：该项目的长期目标是阐明脂筏结构域在晶状体纤维细胞膜中的功能作用，以及它们的相关蛋白，这对保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突相关，如突起、微绒毛、丝状足和板足。脂筏作为信号转导、钙转运和胆固醇转运的蛋白募集平台。在所有物种的晶状体纤维细胞中，大量互锁结构域（以球窝、突起和微绒毛突起的形式）突出到邻近细胞中，增加了细胞膜表面积。由于晶状体是一个维管器官，这种独特的互锁结构域（IDs）的结构配置对于促进细胞之间的分子交换是理想的。在本项目中，我们重点研究了胚胎和成年鸡晶状体id中脂筏的关键脂质和蛋白质成分的鉴定。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是晶状体纤维细胞中的脂筏。目的1通过使用冷冻断裂TEM的filipin细胞化学来验证这一假设的预测，即胆固醇在各种IDs中富集。目的2采用免疫金电镜高压冷冻技术，对IDs中caveolin-1、PMCA、Src激酶和其他关键筏相关蛋白的富集预测进行了验证。目的3通过使用冷冻断裂免疫金复制标记（FRIL）和生物化学方法验证了我们假设的结论，即球窝结构域和平面细胞膜中的cav-1连接蛋白相互作用对晶状体纤维中的间隙连接组装和成熟至关重要。具体来说，我们将利用我们的创新方法，在EM水平上结合filipin细胞化学和FRIL，研究cav-1在胆固醇运输中的作用，以研究在连接组装、成熟和衰老过程中富含胆固醇的间隙连接向无胆固醇的间隙连接的转化。定量分析将用于评估cav-1与含胆固醇间隙连接的关联，因为它们在晶状体中作为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，Aim 4将通过高压冷冻程序确定肌动蛋白分支网络配置，并识别肌动蛋白相关蛋白（例如，Arp2/3， ezrin和paremmin）在形成筏状结构域中的作用。