Gap junctions (GJs) are specializations of the plasma membrane, which link most neighboring animal cells. These junctions contain cell-to-cell channels, providing for the direct, passive exchange of small molecules between cells. An expanding set of evidence indicates that this form of intercellular communication is critical for a wide variety of biological processes, including tissue homeostasis, cellular differentiation, cell signaling and the control of cell growth. Defects or alterations in GJs are thought to play a role in different health-related problems, such as cancerous growth. The present proposal, based on a long-term interest in GJ assembly, describes experiments with reaggregating Novikoff hepatoma cells. This culture system is well-suited to quantitative studies of not only GJ structure and physiology, but also biochemistry, since a GJ protein (a """"""""connexin') has recently been detected and partially characterized in Novikoff cells. Based on our earlier work with these cells, a GJ assembly model has been developed which incorporates self-assembly principles and emphasizes the role of the GJ """"""""formation plaque"""""""". The proposed experiments are designed to evaluate the model and address the following questions: What is the nature and extent of the GJ precursor """"""""pool"""""""" which appears to exist in the plasma membrane? What is the nature of cytoplasmic pools? Since our recent studies with immunological probes indicate that interactions involving connexins are required for GJ assembly, what is the nature of these interactions? Can connexin peptides aid an analysis of GJ assembly? How free are the connexins to diffuse laterally within the plasma membrane? Can connexins be identified in the formation plaque? Is phosphorylation of connexins related to GJ assembly? Other experiments relate more specifically to the regulation of GJ assembly, since cases of enhanced assembly have been documented for Novikoff cells. Our latest experiments indicate that low density lipoprotein (LDL) and its major protein component, apolipoprotein B, can trigger enhanced assembly. What mechanisms are responsible for the enhancement? In addition to these studies of reaggregating Novikoff cells, future work will involve in vitro assembly (i.e., reconstitution of GJ structures). In the Novikoff work, freeze-fracture EM will be used routinely to monitor the development of GJ structures between Novikoff cells. To assess the functional aspects of assembly, the transfer of fluorescent dyes between cells will be analyzed quantitatively, following intracellular injection. Plasma membranes, GJs and GJ proteins will also be isolated and examined in different ways. For example, they will be immunoprecipitated with connexin antibodies to evaluate cellular distribution and phosphorylation levels. Since GJ proteins and, the assembly process display significant conservation, the above approaches should provide valuable insights into the mechanisms of assembly, which operate in all connexin-based GJ systems.
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