Characterization of the lens fiber cell tricellular junctional complex and its dependency on delta-catenin

晶状体纤维细胞三细胞连接复合体的表征及其对δ-连环蛋白的依赖性

• 批准号: 10738883
• 负责人: Timothy F Plageman
• 金额: $ 43.31万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10738883
• 关键词: Adherens Junction; Adhesions; Adhesives; Affinity; Alzheimer' s Disease; Biological Assay; Biology; Cadherins; Cataract; Cell membrane; Cells; Cellular Membrane; Cellular Structures; Cellular biology; Complement; Complex; Cri-du-Chat Syndrome; Critical Thinking; Data; Dependence; Desmosomes; Disease; Environment; Epithelium; Esthesia; Etiology; Genes; Guanosine Triphosphate Phosphohydrolases; Human; Individual; Investigation; Knowledge; Knowledge acquisition; Length; Lens Diseases; Lens Fiber; Link; Mechanics; Microscopy; Modeling; Molecular; Morphology; Mus; Mutant Strains Mice; Mutation; Myopia; Neurosciences Research; Pattern; Physiology; Play; Property; Proteins; Proteomics; Public Health; Resolution; Role; Schizophrenia; Series; Set protein; Signal Transduction; Structure; Study models; Techniques; Testing; Tight Junctions; Time; Tissues; Vertebrates; Vision; afadin; anticancer research; autism spectrum disorder; cancer type; delta protein; delta-catenin; experimental study; fiber cell; follow-up; high resolution imaging; in vivo; insight; lens; loss of function mutation; mechanical force; mechanical load; member; novel; preservation; protein complex; protein function; response; ultra high resolution

Abstract: Lens fiber cells are organized into a regular hexagonal array which promotes the transparency and refractive power of the lens. Junctional adhesion complexes are thought to facilitate this cellular organization but most efforts have focused on proteins and their associated complexes localized within bicellular membranes, where two cell membranes meet. The repeating hexagonal cellular pattern also naturally creates a different type of junction at the confluence of three cells called the tricellular junction which, in other epithelia, is inhabited by a distinct complement of proteins. However, the majority of what is known about these distinctive proteins are part of tricellular tight-junctions (tTJs) while little knowledge exists on the protein complexes that make up tricellular adherens junctions (tAJs) in vertebrates. The tAJ of lens fiber cells are large due to their extreme length and makes them an ideal model for elucidating the biology of this structure. We have recently demonstrated that the cadherin-associated protein δ-catenin is among proteins that specifically localize to the tricellular, but not the bicellular junctions, of lens fiber cells in mice. Furthermore, preliminary proteomic data suggests δ- catenin associates with a distinct set of proteins that include members of desmosomal junctions and GTPase signaling regulators. Importantly, human mutations within the encoding gene of δ-catenin, CTNND2, are associated with both cortical cataracts and high myopia, supporting the notion that both δ-catenin and tricellular junctions are important for lens physiology. However, what functional role δ-catenin plays in the lens or within the tricellular junctional complex of any tissue has not been assessed. In this exploratory proposal, the hypothesis that δ-catenin is required for the normal organization of the tricellular adherens junctional complex of lens fiber cells which preserves lens refraction and transparency will be tested with two aims. In the first aim, the requirement of δ-catenin for lens function will be tested. Additionally, we will perform a series of in vivo and ex vivo interaction assays to test for the presence of hypothesized candidate protein interactions within the tAJ. This analysis will be performed in lens fiber cell lysates and tissue of control or mutant mice lacking functional δ- catenin and followed up with super-resolution fluorescent microscopy to determine tAJ localization. In the second aim, we will test whether the δ-catenin associated protein complex is dependent or independent of the tAJ residing protein afadin and/or mechanical force. Preliminary data indicates that afadin localizes exclusively to tAJs and not bicellular membranes of lens fiber cells and is required for lens fiber cell organization and transparency. The complement of tAJ proteins that depend on afadin to associate with δ-catenin will be determined from proteomic analysis and high-resolution microscopy. Together these experiments will be the first to characterize the function of δ-catenin in the lens and identify the constituent proteins of the tricellular adherens junctional complex of lens fiber cells.

摘要：晶状体纤维细胞组织成规则的六边形阵列，提高了透明度和 晶状体的屈光力被认为有助于这种细胞组织。 但大多数努力都集中在位于双细胞膜内的蛋白质及其相关复合物， 两个细胞膜相遇的地方，重复的六角形细胞图案也自然地产生了不同的类型。 位于三个细胞汇合处的连接处，称为三细胞连接，在其他上皮细胞中，三细胞连接是由 然而，关于这些独特蛋白质的大部分知识都是未知的。 是三细胞紧密连接 (tTJ) 的一部分，而对组成的蛋白质复合物知之甚少 脊椎动物中的三细胞粘附连接 (tAJ) 晶状体纤维细胞的 tAJ 由于其极端长度而很大。 并使它们成为阐明这种结构的生物学的理想模型。 钙粘蛋白相关蛋白 δ-连环蛋白是特异性定位于三细胞的蛋白质之一，但是 不是小鼠晶状体纤维细胞的双细胞连接此外，初步蛋白质组数据表明δ-。 连环蛋白与一组独特的蛋白质相关，其中包括桥粒连接和 GTP 酶的成员 重要的是，δ-连环蛋白编码基因 CTNND2 内的人类突变是。 与皮质白内障和高度近视相关，支持δ-连环蛋白和三细胞 连接对于晶状体生理学很重要但是，δ-连环蛋白在晶状体中或内部发挥什么功能作用。 在此探索性提议中，尚未评估任何组织的三细胞连接复合体的假设。 晶状体纤维三细胞粘附连接复合体的正常组织需要δ-连环蛋白 保留晶状体折射和透明度的细胞将通过两个目标进行测试。 此外，我们还将测试 δ-连环蛋白对晶状体功能的要求。 体内相互作用测定，以测试 tAJ 内是否存在敲击的候选蛋白相互作用。 将在缺乏功能性 δ- 的对照或突变小鼠的晶状体纤维细胞裂解物和组织中进行分析 连环蛋白并随后使用超分辨率荧光显微镜确定 tAJ 的定位。 第二个目标，我们将测试 δ-连环蛋白相关蛋白复合物是否依赖于或独立于 tAJ 驻留在蛋白质 afadin 和/或机械力上。初步数据表明 afadin 专门定位。 是 tAJ 而不是晶状体纤维细胞的双细胞膜，是晶状体纤维细胞组织所必需的， 依赖于 afadin 与 δ-连环蛋白结合的 tAJ 蛋白的补体将是 这些实验将通过蛋白质组分析和高分辨率显微镜来确定。 首先表征了δ-连环蛋白在晶状体中的功能并鉴定了三细胞的组成蛋白 粘附晶状体纤维细胞的连接复合体。