ENHANCED GAP JUNCTION ASSEMBLY CX43 TRAFFICKING

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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