LENS CELL COUPLING--ASSEMBLY AND ROLE OF GAP JUNCTIONS

成熟的晶状体纤维细胞缺乏生物合成细胞器，因此 唯一依赖于与新陈代谢的细胞间的通讯 活跃的晶状体前上皮细胞所需的营养和离子 维持晶状体内环境平衡，防止白内障形成。三 密切相关类型的缝隙连接蛋白(连接蛋白)参与 将晶状体细胞连接成合胞体的分子小于 约1kD：上皮细胞中的连接蛋白43，以及连接蛋白46和 50个(或它们的鸡连接蛋白56和45.6的同源物) 主要存在于纤维细胞中。我已经开发了生化分析方法 缝隙连接组装的主要步骤(连接蛋白寡聚、转运 到质膜，磷酸化，并聚集成GAP 交界处斑块)，并曾使用这些技术研究缝隙 组织培养成纤维细胞的连接形成。第一个目标是 建议的研究是应用和扩展从这方面获得的知识 用于确定如何建立和调节缝隙连接的模型系统 在脊椎动物的晶状体中，可能对晶状体至关重要的过程。 清晰度。这些实验将使用完整的胚胎雏鸡进行 镜片和从它们衍生出来的原始文化，我在其中有 先前研究了连接蛋白43在分子上的表达， 形态和生化水平。需要解决的具体问题 包括：L)不同的晶状体连接蛋白曾经在同一个群体中共组装吗 细胞，潜在地创造新的频道表型？2)什么是 蛋白质(伴侣)和细胞内条件导致 新的内质网后寡聚途径 合成的连接蛋白被组装成半通道(连接蛋白)？ 钙依赖性和非依赖性细胞间黏附的作用是什么 分子(CAM)在植物缝隙连接建立和维持中的作用 以及是否有证据表明连接蛋白之间的信号转导 摄像头呢？这项提议的第二个目标是设计一种突变体 将与之共同组装的晶状体纤维连接蛋白(Cx46)的形式，以及 抑制野生型晶状体连接蛋白的通道形成功能。是这样的 一种显性-负性方法被成功地用来扰动GAP 非洲爪哇早期胚胎中连接介导的细胞间通讯 (Paul等人，1993)。最终，这个cx46突变体将在 晶状体选择性αA结晶蛋白控制下的转基因小鼠 启动子提供定义体内作用的第一个分子证据 晶状体发育、生理学和白内障病理学中缝隙连接的研究。

Mature lens fiber cells lack biosynthetic organelles and are therefore uniquely dependent on intercellular communication with the metabolically active anterior lens epithelial cells for the nutrients and ions required to maintain lens homeostasis and prevent cataract formation. Three closely related types of gap junction proteins (connexins) participate in joining lens cells into a syncytium with respect to molecules smaller than approximately 1 kD: connexin43 in epithelial cells, and connexins 46 and 50 (or their chick homologues connexins 56 and 45.6) which are found predominantly in fiber cells. I have developed biochemical assays for the major steps in gap junction assembly (connexin oligomerization, transport to the plasma membrane, phosphorylation, and clustering into gap junctional plaques) and have previously used these techniques to study gap junction formation in tissue culture fibroblasts. The first goal of the proposed studies is to apply and extend the knowledge obtained from this model system to determine how gap junctions are established and regulated in the vertebrate lens, processes that are likely to be essential for lens clarity. These experiments will be performed using intact embryonic chick lenses and primary cultures derived from them, systems in which I have previously characterized connexin43 expression on a molecular, morphological, and biochemical level. Specific issues to be addressed include: l) do the different lens connexins ever coassemble in the same cell, potentially creating new channel phenotypes? 2) what are the proteins (chaperones) and intracellular conditions responsible for the novel, post-endoplasmic reticulum oligomerization pathway by which newly synthesized connexins are assembled into half-channels (connexins)? 3) what are the roles of Ca++-dependent and -independent cell-cell adhesion molecules (CAMs) in the establishment and maintenance of gap junctions in the lens, and is there evidence for signal transduction between connexins and CAMs? The second objective of this proposal is to design a mutant form of a lens fiber connexin (cx46) that will coassemble with, and inhibit the channel-forming function of, wild-type lens connexins. Such a dominant-negative approach has been successfully used to perturb gap junction-mediated intercellular communication in early Xenopus embryos (Paul et al., 1993). Ultimately, this cx46 mutant would be expressed in transgenic mice under the control of the lens-selective alphaA crystallin promoter to provide the first molecular evidence defining the in vivo role of gap junctions in lens development, physiology, and cataract pathology.