Grant: Discovery Grants Program - Individual

It is a generalrequirement for normal cell and tissue function that adjacent cells communicatedirectly with each other through special channels called gap junctions. Ourlaboratory has long been interested in examining the functional role of thesechannels as they serve to exchange small signaling and regulatory moleculesbetween cells and also to function as hemichannels to release molecules outsidethe cell. These unique, large-pore channels are formed from a huge family ofproteins (n=20 in mice) called connexins (Cx). For reasons that we poorlyunderstand, many cell types need to produce multiple members of the connexinfamily and make gap junction channels from combinations of expressed connexins.This phenomena is most evident in the epidermis of the skin where at least 7connexins (called keratinocyte connexins) are expressed. In the epidermis,keratinocytes proceed to differentiation and enter programmed cell deathresulting in the epidermis renewing approximately every 2 weeks. This study isdesigned to determine the life cycle of keratinocyte connexins when co-expressedin the same keratinocytes. It is our hypothesis that keratinocyte connexins engagetrafficking routes and regulatory assembly events that often differ from Cx43(a major keratinocyte connexin), resulting in the formation of distinctchannels that are required for proper epithelial cell function. Thesestudies are particularly important as newly synthesized connexins may betargeted to; a) plasma membranesdevoid of gap junctions where they function as hemichannels, b) gap junctional complexes where they participatein traditional gap junctional intercellular communication or c) mitochondrial inner membranes. Our studies will be performedin rat keratinocytes that have the ability to differentiate into cornifiedepidermis when grown at a liquid/air interface. Both untagged connexins andconnexins that are tagged with fluorescent proteins will be used as this allowsfor the assessment of connexin life-cycles in both fixed and living cells. Wewill employ pharmacological approaches and fluorescent bleaching strategies to simultaneouslydetermine the dynamic properties of multiple connexins and dissect the lifecycle and assembly characteristics of each connexin. In some cases, we expectthat connexins will take up residence in the mitochondria where they mayregulate keratinocytes as they proceed toward programmed cell death. Once weresolve the life cycle of each keratinocyte connexin and determine theirprimary site of residence in living keratinocytes and in organotypic epidermiswe will determine how these events regulate keratinocyte function and differentiation. Significance:   Our studies will provide fundamental insightsinto the localization and function of channels composed of different connexin