Multiple connexin trafficking and sorting in live cells

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It is a general requirement for normal cell and tissue function that adjacent cells communicate directly with each other through special channels called gap junctions. Our laboratory has long been interested in examining the functional role of these channels as they serve to exchange small signaling and regulatory molecules between cells and also to function as hemichannels to release molecules outside the cell. These unique, large-pore channels are formed from a huge family of proteins (n=20 in mice) called connexins (Cx). For reasons that we poorly understand, many cell types need to produce multiple members of the connexin family and make gap junction channels from combinations of expressed connexins. This phenomena is most evident in the epidermis of the skin where at least 7 connexins (called keratinocyte connexins) are expressed. In the epidermis, keratinocytes proceed to differentiation and enter programmed cell death resulting in the epidermis renewing approximately every 2 weeks. This study is designed to determine the life cycle of keratinocyte connexins when co-expressed in the same keratinocytes. It is our hypothesis that keratinocyte connexins engage trafficking routes and regulatory assembly events that often differ from Cx43 (a major keratinocyte connexin), resulting in the formation of distinct channels that are required for proper epithelial cell function. These studies are particularly important as newly synthesized connexins may be targeted to; a) plasma membranes devoid of gap junctions where they function as hemichannels, b) gap junctional complexes where they participate in traditional gap junctional intercellular communication or c) mitochondrial inner membranes. Our studies will be performed in rat keratinocytes that have the ability to differentiate into cornified epidermis when grown at a liquid/air interface. Both untagged connexins and connexins that are tagged with fluorescent proteins will be used as this allows for the assessment of connexin life-cycles in both fixed and living cells. We will employ pharmacological approaches and fluorescent bleaching strategies to simultaneously determine the dynamic properties of multiple connexins and dissect the life cycle and assembly characteristics of each connexin. In some cases, we expect that connexins will take up residence in the mitochondria where they may regulate keratinocytes as they proceed toward programmed cell death. Once we resolve the life cycle of each keratinocyte connexin and determine their primary site of residence in living keratinocytes and in organotypic epidermis we will determine how these events regulate keratinocyte function and differentiation. Significance:  Our studies will provide fundamental insights into the localization and function of channels composed of different connexins in epidermal renewal.