Multiple connexin trafficking and sorting in live cells

活细胞中的多重连接蛋白运输和分类

• 批准号: RGPIN-2015-04277
• 负责人: Laird, Dale
• 金额: $ 3.28万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=689038
• 关键词: Multiple; connexin; trafficking; sorting; live

**************** ****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:

**************** ****这是正常细胞和组织功能的一般要求，相邻细胞通过称为间隙连接的特殊通道直接相互通信。长期以来，我们的实验室一直对研究这些通道的功能作用很感兴趣，因为它们在细胞之间交换小信号和调节分子，并作为半通道将分子释放到细胞外。这些独特的大孔通道是由一个叫做连接蛋白（Cx）的巨大蛋白质家族（小鼠中n=20）形成的。由于我们不太了解的原因，许多细胞类型需要产生多个连接蛋白家族成员，并通过表达的连接蛋白组合形成间隙连接通道。这种现象在皮肤表皮最明显，至少有7*连接蛋白（称为角化细胞连接蛋白）表达。在表皮中，角质形成细胞进行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。本研究旨在确定角化细胞连接蛋白在相同角化细胞中共表达时的生命周期。我们的假设是，角质细胞连接蛋白参与的运输路线和调节组装事件通常不同于Cx43*（一种主要的角质细胞连接蛋白），导致形成不同的通道，这是上皮细胞正常功能所必需的。这些研究尤其重要，因为新合成的连接蛋白可能针对；A)质膜缺乏作为半通道的间隙连接，b)间隙连接复合物参与传统的间隙连接细胞间通讯，或c)线粒体内膜。我们的研究将在大鼠角质形成细胞中进行，这些细胞在液体/空气界面生长时具有分化成角化表皮的能力。将使用未标记的连接蛋白和用荧光蛋白标记的连接蛋白，因为这允许在固定细胞和活细胞中评估连接蛋白的生命周期。我们将采用药理学方法和荧光漂白策略同时确定多个连接蛋白的动态特性，并剖析每个连接蛋白的生命周期和组装特性。在某些情况下，我们预计连接蛋白将在线粒体中居住，在那里它们可能调节角质形成细胞走向程序性细胞死亡。一旦我们解决了每个角化细胞连接蛋白的生命周期，并确定了它们在活的角化细胞和器官型表皮中的主要居住位置，我们将确定这些事件如何调节角化细胞的功能和分化。