GAP JUNCTION ASSEMBLY--MECHANISMS AND REGULATIONS

缝隙连接(GJS)是质膜的特化，它 连接大多数邻近的动物细胞。这些连接包含细胞与细胞之间 通道，提供小分子的直接、被动交换 在细胞之间。越来越多的证据表明，这种形式的 细胞间的通讯对于各种各样的生物来说是至关重要的 过程，包括组织内稳态、细胞分化、细胞 信号和细胞生长的控制。GJS的缺陷或改变 被认为在不同的与健康相关的问题中发挥作用，例如 癌变的生长。目前的建议，是基于对 GJ组装，描述了重新聚集Novikoff肝癌的实验 细胞。这种文化体系非常适合于对NOT的定量研究 不仅是GJ的结构和生理，还有生物化学，因为GJ 蛋白质(一种“连接蛋白”)最近被检测到并部分地 以诺维科夫细胞为特征。基于我们之前对这些内容的研究 Cells，开发了GJ装配模型，该模型结合了 自组装原理和强调GJ“形成”的作用 拟议的实验旨在评估该模型和 回答以下问题：联合司法的性质和范围是什么 质膜中似乎存在的前体“池”？是什么 细胞质池的性质？自从我们最近对 免疫探针表明，涉及连接蛋白的相互作用是 GJ组装所需的，这些相互作用的性质是什么？能 连接蛋白多肽有助于分析GJ组装？他们有多自由？ 连接蛋白在质膜内横向扩散？CaN连接蛋白 在队形斑块中被辨认出来？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到 对GJ组装的监管，因为增强组装的情况已经 关于诺维科夫牢房的记录。我们最新的实验表明，低 密度脂蛋白及其主要蛋白质组分载脂蛋白 B，可以触发增强的组装。是什么机制导致了 增强版？除了这些关于重新聚合Novikoff的研究 细胞，未来的工作将涉及体外组装(即重建 GJ结构)。在Novikoff工作中，将使用冷冻骨折EM 常规监测诺维科夫与诺维科夫之间GJ结构的发展 细胞。为了评估组装的功能方面，将 细胞间的荧光染料将进行定量分析，如下 细胞内注射。质膜、GJS和GJ蛋白也将 以不同的方式被隔离和检查。例如，它们将是 缝隙连接蛋白抗体免疫沉淀法评价细胞 分布和磷酸化水平。由于GJ蛋白和 装配工艺显示出显著的节约，以上方法 应该为组装的机制提供有价值的见解，这些机制 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.