LENS CELL COUPLING--ASSEMBLY AND ROLE OF GAP JUNCTIONS

成熟的透镜纤维细胞缺乏生物合成细胞器，因此 独特地依赖于细胞间的通讯， 活跃的前透镜上皮细胞提供所需的营养和离子 以维持透镜体内平衡并防止白内障形成。 三 密切相关的间隙连接蛋白（连接蛋白）参与 将透镜细胞连接成合胞体，其分子小于 约1 kD：上皮细胞中的连接蛋白43，以及连接蛋白46和 50（或它们的鸡同源物连接蛋白56和45.6），它们被发现于 主要存在于纤维细胞中。我已经开发出了生化检测方法， 间隙连接组装的主要步骤（连接蛋白寡聚化、转运 到质膜，磷酸化，并聚集成间隙 交界斑块），并且之前曾使用这些技术来研究间隙 组织培养成纤维细胞中连接形成。 的第一个进球 建议的研究是应用和扩展从中获得的知识， 模型系统，以确定间隙连接是如何建立和调节的 在脊椎动物的透镜中，可能对透镜至关重要的过程 清楚起见 这些实验将使用完整的鸡胚进行 镜头和主要文化来源于他们，系统中，我有 先前表征的连接蛋白43在分子上的表达， 形态学和生化水平。 有待解决的具体问题 包括：l）不同的透镜连接蛋白是否曾经共同组装在相同的 细胞，可能创造新的通道表型？2)有哪些 蛋白质（伴侣）和细胞内条件负责 一种新的内质网后寡聚化途径， 合成的连接蛋白组装成半通道（连接蛋白）？第三章 钙依赖性和非依赖性细胞间粘附的作用是什么 分子（CAM）在建立和维持间隙连接中的作用 透镜，是否有证据表明连接蛋白之间的信号转导 CAMs？ 这个计划的第二个目标是设计一个突变体 透镜形式的纤维连接蛋白（CX 46），其将与 抑制野生型透镜连接蛋白的通道形成功能。 等 显性负的方法已成功地用于扰动间隙 连接介导早期爪蟾胚胎细胞间通讯 (Paul例如，1993年）。 最终，这种cx46突变体将在 晶状体选择性α A晶状体蛋白控制下的转基因小鼠 启动子提供了定义体内作用的第一个分子证据 缝隙连接在透镜发育、生理学和白内障病理学中的作用。

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.