ENHANCED GAP JUNCTION ASSEMBLY CX43 TRAFFICKING

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。该项目重点研究了camp介导的培养成纤维细胞间隙连接（GJ）组装的增强，这需要连接蛋白43 （Cx43）的细胞内运输。我们正在使用加州大学圣地亚哥分校研究人员开发的四胱氨酸标记和检测方法研究Cx43的贩运。这包括对活细胞的荧光显微镜，以及对这些细胞的厚切片和层析成像的相关电子断层扫描。与Gina Sosinsky博士及其同事合作，将解决两个具体方面的问题：a)对贩运中间体的微管依赖性运输的分析，以响应cAMP； b)非连接半通道被内噬，然后以一种受调节的方式（例如，在cAMP的控制下）再循环到质膜的假设的测试。我们目前使用Cx43-GFP构建体进行常规荧光工作。最近，我们发现Cx43- gfp在成纤维细胞中与Cx43共表达导致与cAMP a的组装增加了4倍。微管依赖运输我们目前正在表征含有Cx43- gfp的膜的细胞内运动，并将我们的经验应用于Cx43-四胱氨酸构建物（Cx43- cys4）。这包括在微管抑制剂诺可达唑（nocodazole）存在和不存在的情况下，对录像中的运动速率进行分析。我们想确定这些运动是否沿着微管排列，可能是双向运输。我们想要确定cAMP是否会影响贩运率和/或贩运活动水平，因为cAMP治疗将组装提高了至少2至3倍。B.内吞作用和再循环我们对活细胞中使用tc标记和荧光转铁蛋白监测转铁蛋白受体摄取的cx43半通道的假设内吞作用感兴趣。抗体标记将有助于在固定材料中识别不同的膜系统（例如，使用针对不同兔蛋白的抗体）。EM研究将确定含有Cx43-TC的内吞囊泡（也标记有金标记的转铁蛋白或铁蛋白）是否被包被（如网格蛋白），以及含有Cx43-TC证据的早期内体腔室的性质。在可能的再循环研究中，我们寻找Cx43-TC在中心周围再循环中心和高尔基体（帮助分类内吞作用）中的证据，以及在据报道显示再循环受体的早期核内体的管状延伸中

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project focuses on the cAMP-mediated enhancement of gap junction (GJ) assembly in cultured fibroblasts, which requires the intracellular trafficking of connexin43 (Cx43). We are studying the trafficking of Cx43, by using the tetracysteine tagging and detection methods developed by investigators at UCSD. This includes fluorescence microscopy on living cells, as well as correlated electron tomography on thick sections and tomographic imaging of these cells. Two specific aspects will be addressed, in collaboration with Dr. Gina Sosinsky and colleagues: a) an analysis of the microtubule-dependent transport of trafficking intermediates, in response to both cAMP and b) a testing of the hypothesis that non-junctional hemichannels are endocytosed and then recycled back to the plasma membrane in a regulated manner (e.g., under the control of cAMP). We are presently using Cx43-GFP constructs for routine fluorescence work. Recently, we have found that Cx43-GFP co-expressed in fibroblasts with Cx43 resulted in a 4-fold increase in assembly with cAMP A. Microtubule-Dependent Trafficking We are presently characterizing the intracellular movement of membranes containing Cx43-GFP and will apply our experiences to Cx43-tetracysteine constructs (Cx43-Cys4). This includes analysis of rates of movement in video recordings, in the presence and absence of nocodazole, a microtubule inhibitor. We want to determine whether these movements are aligned along microtubules, with possible bi-directional trafficking. We want to determine whether cAMP affects the rate of trafficking and/or the level of trafficking activity, given that cAMP treatment enhances assembly at least 2- to 3-fold. B. Endocytosis and Recycling We are interested in the postulated endocytosis of Cx43-hemichannels in living cells with the TC-tagging and with the use of fluorescent transferrin to monitor uptake of the transferrin receptor. Antibody markers will help identify different membrane systems in fixed material (e.g., using antibodies for different rab proteins). EM studies will determine whether endocytotic vesicles with Cx43-TC (marked also with gold-labeled transferrin or ferritin) are coated (e.g., with clathrin) and the nature of early endosomal compartments with evidence of Cx43-TC. In studies of possible recycling, we look for evidence of Cx43-TC in the pericentriolar recycling center vs. the Golgi (to help classify endocytosis) and in the tubular extensions from early endosomes that are reported to display recycling receptors