Multiple connexin trafficking and sorting in live cells

点击翻译按钮获取中文摘要

**************** ****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.  ************