ENHANCED GAP JUNCTION ASSEMBLY CX43 TRAFFICKING

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定是研究者机构。本项目主要研究cAMP介导的成纤维细胞间隙连接（gap junction，GJ）组装的增强，这需要连接蛋白43（connexin 43，Cx43）的胞内运输。我们正在研究Cx43的贩运，通过使用UCSD研究人员开发的四半胱氨酸标记和检测方法。这包括活细胞上的荧光显微镜，以及厚切片上的相关电子断层扫描和这些细胞的断层成像。与Gina Sosinsky博士及其同事合作，将解决两个具体方面：a）响应于cAMP和B的运输中间体的微管依赖性运输的分析b）对非连接半通道被内吞然后以调节的方式再循环回质膜的假设的测试（例如，在cAMP的控制下）。我们目前正在使用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4倍。微管依赖性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Cx43-四半胱氨酸构建体（Cx43-Cys 4）。这包括在存在和不存在诺考达唑（一种微管抑制剂）的情况下分析视频记录中的运动速率。我们想确定这些运动是否沿着沿着微管排列，可能是双向运输。我们想确定cAMP是否影响贩运率和/或贩运活动的水平，因为cAMP处理使组装增强至少2至3倍。B。内吞作用和再循环我们感兴趣的是假设的内吞作用的Cx43-半通道在活细胞与TC-标签和使用荧光转铁蛋白监测摄取的转铁蛋白受体。抗体标记物将有助于鉴定固定材料中的不同膜系统（例如，使用针对不同RAB蛋白的抗体）。EM研究将确定具有Cx43-TC的内吞囊泡（也用金标记的转铁蛋白或铁蛋白标记）是否被包被（例如，与网格蛋白）和早期内体区室的性质与Cx43-TC的证据。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与高尔基体（以帮助分类内吞作用）以及据报道显示再循环受体的早期内体的管状延伸中的证据

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