Gap Junction Assembly: Mechanisms and Regulation

动物细胞间广泛存在的一种通讯方式是通过细胞直接、被动地交换小分子。细胞通道。这些通道由脊椎动物的“连接蛋白”组成，聚集形成间隙连接（GJs），在电信号的传递、细胞生长的控制和各种器官系统的发育中发挥重要作用。GJ通讯的重要性反映在与连接缺陷和/或连接蛋白突变相关的疾病状态的数量上。鉴于连接素的半衰期短，有必要建立有效的机制来定性和定量地调节gj的组装。本实验室的目标是了解G3组装过程及其调节。我们的研究方法是基于一套完整的结构、功能和分子方法应用于培养细胞。我们相信这将提供对GJ组装及其规则的广泛理解。本文提出的研究将集中于增强GJ组装，这是一个由cAMP介导的过程，依赖于细胞内Cx43的运输。虽然连接蛋白运输是细胞通信的一个鲜为人知的方面，但它可能在细胞生理学和病理学中都很重要。我们将解决以下三个目标：目标# 1：确定微管依赖性Cx43-化学机制如何受到cAMP、细胞接触和特异性Cx43突变的影响。目标2：确定是否没有。连接质膜半通道被内吞和再循环，以及这是否受到camp依赖机制的调节。目的3：确定具有cx43半通道的膜如何沿着微管运输，并对cAMP和细胞接触的增加做出反应。GJ组装的功能（染料转移）和结构（冷冻断裂电镜）分析将用于检测转染野生型Cx43或不同突变形式的“敲除”小鼠的成纤维细胞。特异性Cx43抗体抑制质膜（PM）半通道后的“染料摄取恢复”，以及non的生物素化。连接，PM中的Cx43，将用于测量向PM的半通道的输送。为了分析活细胞中的转运，我们将使用Cx43-GFP（绿色荧光蛋白）嵌合体。我们还将采用新开发的方法用四胱氨酸标记Cx43，用于活细胞的荧光研究和EM水平的研究。最后，“下拉”实验将鉴定与cAMP相关的Cx43蛋白，并使用各种生化技术研究成纤维细胞中野生型和突变型Cx43。网站性能 ======================================== 节结束 ===========================================

EXCEEDTHESPACEPROVIDED, A widespread means of communication between animal cells involves the direct, passive exchange of small molecules via cell.to-cell channels. These channels, comprised of"connexins" in vertebrates, are aggregated to form gapjunctions (GJs) and play a significant role in the transmission of electrical signals, the control of cell growth and the development of various organ systems. The importance of GJ communication is reflected in the number of disease states linked to junctional deficiences and/or connexin mutations. Given the short half-lives of connexins, it is necessary to have efficient and effective mechanisms for regulating the assembly of GJs, both qualitatively and quantitatively. The goal of this laboratory is to understand the process of G3 assembly and its regulation. Our research approach is based on an integrated set of structural, functional, and molecular methods applied to cultured cells. We believe this will provide for a broad understanding of GJ assembly and its regulation. The studies proposed here will focus on enhanced GJ assembly, a process that is mediated by cAMP and that relies on the intracellular trafficking of Cx43. Although connexin trafficking represents a poorly understood aspect of cell communication, it it likely to be important in both cellular physiology and pathology. We will address the following three aims: Aim # 1: Determine how the micrutubule.depondent detivepj at Cx43-hemichanneis to the plasma membrane is influenced by cAMP, cell contact and specific Cx43 mutations. Aim #2: Determine whether non.junctional, plasma membrane hemichannels are endocytosed and recycled and whether this is regulated by a cAMP-dependent mechanism. Aim #3: Determine how membranes with Cx43-hemichannels are transported along microtubules and respond to increases in both cAMP and cell contact. Both functional (dye transfer) and structural (freeze-fracture EM) assays for GJ assembly will be used to examine flbroblasts from "knock out" mice transfectod with wildtype Cx43 or different mutant forms. The "restoration of dye uptake" following the inhibition of plasma membrane (PM) hemichannels with specific Cx43 antibodies, as well as the biotinylation of non.junctional, Cx43 in the PM, will be used to measure the delivery of hemichannels to the PM. To analyze transport in living ceils, we will use Cx43-GFP (green fluorescence protein) chimeras. We will also employ newly developed methods tar tagging Cx43 with tetracysteine for fluorescence studies in living cells and for studies at the EM level. Finally, "pull-down" experiments will identify proteins associating with Cx43, in response to cAMP, and various biochemical techniques will be used to study wild-type and mutant Cx43 in flbroblasts. PERFORMANCE SITE ========================================Section End===========================================