GAP JUNCTION ASSEMBLY--MECHANISMS AND REGULATION

间隙连接（GJ）是质膜的特化，其 连接大多数邻近的动物细胞。 这些连接包含了细胞与细胞之间 通道，提供小分子的直接、被动交换 细胞之间。 越来越多的证据表明， 细胞间通讯对于多种生物学过程是至关重要的， 过程，包括组织内稳态、细胞分化、细胞分化、 信号传导和细胞生长的控制。 GJ的缺陷或变更 被认为在不同的健康相关问题中发挥作用，例如 癌症生长 目前的建议，基于长期利益， GJ组装，描述了重新聚集Novikoff肝癌的实验 细胞 这种培养系统非常适合于非生物学的定量研究。 不仅GJ结构和生理学，而且生物化学，因为GJ 蛋白质（一种“连接蛋白”）最近被检测到， 在Novikoff细胞中的特征。 基于我们之前对这些 细胞，一个GJ组装模型已经开发，其中包括 自组装原理，并强调了GJ“形成的作用 斑块”。 所提出的实验旨在评估该模型， 解决以下问题：GJ的性质和范围是什么 前体“池”似乎存在于质膜？是什么 细胞质池的本质由于我们最近的研究， 免疫学探针表明，涉及连接蛋白的相互作用， GJ组装所需的，这些相互作用的性质是什么？可以 连接蛋白肽有助于GJ组装的分析？有多自由 连接蛋白在质膜内横向扩散？Can连接蛋白 在地层斑块中被识别？是连接蛋白的磷酸化 与GJ组装有关？其他实验更具体地涉及 GJ组装的调节，因为增强组装的情况已经 记录了Novikoff细胞。 我们最新的实验表明， 密度脂蛋白（LDL）及其主要蛋白质成分载脂蛋白 B，可以触发增强组装。 哪些机制负责 增强？除了这些重新聚集诺维科夫的研究 细胞，未来的工作将涉及体外组装（即，复溶 GJ结构）。 在Novikoff的工作中，将使用冷冻断裂EM 定期监测Novikoff之间GJ结构的发展 细胞 为了评估组装的功能方面，转移 细胞之间的荧光染料将被定量分析， 细胞内注射 质膜、GJ和GJ蛋白也将 以不同的方式进行隔离和检查。 例如，他们将 用连接蛋白抗体进行免疫沉淀，以评估细胞 分布和磷酸化水平。 由于GJ蛋白和， 装配过程中显示出明显的节约，上述方法 应该提供宝贵的见解，以大会的机制， 在所有基于连接蛋白的GJ系统中运行。

Gap junctions (GJs) are specializations of the plasma membrane, which link most neighboring animal cells. These junctions contain cell-to-cell channels, providing for the direct, passive exchange of small molecules between cells. An expanding set of evidence indicates that this form of intercellular communication is critical for a wide variety of biological processes, including tissue homeostasis, cellular differentiation, cell signaling and the control of cell growth. Defects or alterations in GJs are thought to play a role in different health-related problems, such as cancerous growth. The present proposal, based on a long-term interest in GJ assembly, describes experiments with reaggregating Novikoff hepatoma cells. This culture system is well-suited to quantitative studies of not only GJ structure and physiology, but also biochemistry, since a GJ protein (a "connexin') has recently been detected and partially characterized in Novikoff cells. Based on our earlier work with these cells, a GJ assembly model has been developed which incorporates self-assembly principles and emphasizes the role of the GJ "formation plaque". The proposed experiments are designed to evaluate the model and address the following questions: What is the nature and extent of the GJ precursor "pool" which appears to exist in the plasma membrane? What is the nature of cytoplasmic pools? Since our recent studies with immunological probes indicate that interactions involving connexins are required for GJ assembly, what is the nature of these interactions? Can connexin peptides aid an analysis of GJ assembly? How free are the connexins to diffuse laterally within the plasma membrane? Can connexins be identified in the formation plaque? Is phosphorylation of connexins related to GJ assembly? Other experiments relate more specifically to the regulation of GJ assembly, since cases of enhanced assembly have been documented for Novikoff cells. Our latest experiments indicate that low density lipoprotein (LDL) and its major protein component, apolipoprotein B, can trigger enhanced assembly. What mechanisms are responsible for the enhancement? In addition to these studies of reaggregating Novikoff cells, future work will involve in vitro assembly (i.e., reconstitution of GJ structures). In the Novikoff work, freeze-fracture EM will be used routinely to monitor the development of GJ structures between Novikoff cells. To assess the functional aspects of assembly, the transfer of fluorescent dyes between cells will be analyzed quantitatively, following intracellular injection. Plasma membranes, GJs and GJ proteins will also be isolated and examined in different ways. For example, they will be immunoprecipitated with connexin antibodies to evaluate cellular distribution and phosphorylation levels. Since GJ proteins and, the assembly process display significant conservation, the above approaches should provide valuable insights into the mechanisms of assembly, which operate in all connexin-based GJ systems.