Characterization of proteins and cellular pathways involved in regulation of gap junction mediated cell-cell communication and physical coupling

Gap Junction (GJ) channels bridge apposing plasma membranes (PMs) to provide direct cell-to-cell communication between adjacent cells via the sharing of ions and small molecules. Each channel consists of two hemichannels (connexons), docked to each other in the extracellular space. Once joined the connexons cannot separate under physiological conditions, thus providing a strong physical bond between the adjoining PMs. Gap junction intercellular communication (GJIC) is vital to all aspects of cellular life and can be modulated under both physiological and pathological conditions. Rapid turnover of GJ proteins suggests that beyond opening and closing of individual channels, regulation of GJIC can be achieved by altering the number of available GJ channels in the PMs. Internalization of GJs is likely to provide rapid permanent or transient uncoupling under conditions when decreased communication and increased cell migration are favorable. Our spatiotemporal investigations of GJ degradation in living cells demonstrate that entire or large portions of GJs are internalized as double-membrane structures, resulting in the formation of cytoplasmic double-membrane vesicles (sometimes termed annular gap junctions [AGJs]). Internalization occurs preferentially into one of the connected cells, suggesting a highly regulated endocytic process. Early ultrastructural studies revealed such cytoplasmic double-membrane GJ vesicles that were hypothesized to represent early intermediates of internalized GJs. Detection of a clathrin-like bristle-coat on AGJs and our hypotonic-medium-based inhibition of GJ internalization suggested that GJ internalization might occur by a clathrin-mediated endocytic (CME) process. Moreover, our EM analyses identified numerous AGJs surrounded by membranous cisternae suggesting the involvement of autophagy in AGJ degradation.;In the dissertation work presented here, I utilized RNAi technology, pharmacological agents and molecular and biochemical techniques to demonstrate that GJs are indeed internalized via a clathrin-dependent mechanism, and that internalized GJ vesicles are degraded by autophagy. I found that the coat protein, clathrin, together with the clathrin adaptors, AP-2 and Dab2, and the GTPase dynamin mediate the endocytosis of GJ plaques. Additionally, I identified autophagy as the cellular pathway that isolates and degrades internalized GJs.