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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=656383
• 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:  Our studies will provide fundamental insights*into the localization and function of channels composed of different connexins*in epidermal renewal.  ************

** 正常细胞和组织功能的一般要求是相邻细胞通过称为间隙连接的特殊通道直接相互通信。我们的实验室长期以来一直对研究这些通道的功能作用感兴趣，因为它们用于在细胞之间交换小的信号和调节分子，并且还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）的蛋白质大家族（在小鼠中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连接蛋白家族的多个成员，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，其中表达至少7种连接蛋白（称为角质形成细胞连接蛋白）。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。本研究旨在确定角质细胞连接蛋白在同一角质细胞中共表达时的生命周期。我们的假设是，角质形成细胞连接蛋白参与 * 运输途径和调节组装事件，通常不同于Cx43*（一种主要的角质形成细胞连接蛋白），导致形成不同的 * 通道，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靶向：a）缺乏间隙连接的质膜 *，它们在那里起半通道的作用，B）间隙连接复合物，它们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，当在液体/空气界面生长时，角质形成细胞具有分化成表皮的能力。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，同时确定多种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征。在某些情况下，我们预期连接蛋白会驻留在线粒体中，当角质形成细胞走向程序性细胞死亡时，它们可能会调节角质形成细胞。一旦我们解决了每个角质形成细胞连接蛋白的生命周期，并确定了它们在活的角质形成细胞和器官型表皮中的主要驻留位点，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。重要性：我们的研究将为表皮更新中由不同连接蛋白组成的通道的定位和功能提供基本的见解。