Gap Junction Assembly: Mechanisms and Regulation

描述（由申请人提供）：动物细胞之间广泛的通讯方式涉及通过细胞直接、被动交换小分子。到细胞的通道。这些通道由脊椎动物中的"连接蛋白"组成，聚集形成间隙连接（GJ），并在电信号的传递、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通信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中。鉴于连接蛋白的半衰期短，有必要具有高效和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组装过程及其调控。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，这将提供一个广泛的理解GJ组装及其监管。这里提出的研究将集中在增强GJ组装，这是一个由cAMP介导的过程，依赖于Cx43的细胞内运输。虽然连接蛋白的运输代表了一个知之甚少的细胞通讯方面，它可能是重要的细胞生理学和病理学。我们将解决以下三个目标：目标#1：确定如何通过cAMP、细胞接触和特异性Cx43突变影响在Cx43半通道处微管依赖性递送至质膜。目标二：确定非连接性质膜半通道是否被内吞和再循环，以及这是否受cAMP依赖性机制的调节。目的#3：确定具有Cx43-半通道的膜如何沿沿着转运并对cAMP和细胞接触的增加做出响应。GJ组装的功能（染料转移）和结构（冷冻断裂EM）测定将用于检查用野生型Cx43或不同突变形式转染的"敲除"小鼠的成纤维细胞。用特异性Cx43抗体抑制质膜（PM）半通道后的"染料摄取的恢复"以及PM中非连接Cx43的生物素化将用于测量半通道向PM的递送。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焦油标记Cx43与四半胱氨酸在活细胞中的荧光研究和EM水平的研究。最后，“下拉”实验将确定与Cx43相关的蛋白质，响应cAMP，各种生物化学技术将用于研究成纤维细胞中的野生型和突变型Cx43。

DESCRIPTION (provided by applicant): 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 gap junctions (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 deficiencies 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 likely to be important in both cellular physiology and pathology. We will address the following three aims: Aim # 1: Determine how the microtubule dependent delivery at Cx43-hemichannels to the plasma membrane is influenced by cAMP; cell contact and specific Cx43 mutations. Aim #2: Determine whether nonjunctional, 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 fibroblasts from "knock out" mice transfected with wild type 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 nonjunctional, 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 fibroblasts.