Multiple connexin trafficking and sorting in live cells

是一般 正常细胞和组织功能需要相邻细胞进行通信 通过称为缝隙连接的特殊通道直接相互连接。我们 长期以来，实验室一直有兴趣研究这些功能的作用， 通道，因为它们用于交换小信号和调节分子 同时也起到半通道的作用， 牢房这些独特的大孔通道是由一个庞大的家庭， 蛋白质（n=20小鼠）称为连接蛋白（Cx）。因为我们很难 许多细胞类型需要产生连接蛋白的多个成员 家族并从表达的连接蛋白的组合中形成间隙连接通道。 这种现象在皮肤的表皮中最明显，其中至少7 表达连接蛋白（称为角质细胞连接蛋白）。在表皮中， 角质形成细胞继续分化并进入程序性细胞死亡 导致表皮大约每2周更新一次。本研究 旨在确定角质细胞连接蛋白共表达时的生命周期 在相同的角质形成细胞中。我们的假设是角质细胞连接蛋白参与 通常与Cx43不同的贩运途径和调节组装事件 （一种主要的角质细胞连接蛋白），导致形成不同的 这些通道是正常上皮细胞功能所必需的。这些 研究特别重要，因为新合成的连接蛋白可能是 靶向; a）质膜 缺乏间隙连接，其中它们起半通道的作用，B）间隙连接复合物，其中它们参与 在传统的间隙连接细胞间通讯中或c）线粒体内膜中。我们的研究将在 在具有分化成角质形成细胞的能力的大鼠角质形成细胞中， 当在液体/空气界面处生长时表皮。未标记的连接蛋白和 将使用用荧光蛋白标记的连接蛋白 用于评估固定和活细胞中的连接蛋白生命周期。我们 将采用药理学方法和荧光漂白策略， 确定多个连接蛋白的动力学特性， 每个连接蛋白的循环和组装特征。在某些情况下，我们希望 连接蛋白将在线粒体中定居， 调节角质形成细胞，因为它们进行程序性细胞死亡。一旦我们 解析每种角质细胞连接蛋白的生命周期， 在活的角质形成细胞和器官型表皮中的主要驻留部位 我们将确定这些事件如何调节角质形成细胞的功能和分化。 意义：我们的研究将提供基本的见解 由不同连接蛋白组成的通道的定位和功能 在表皮更新中。

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.