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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=612875
• 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.

这是一位将军 相邻细胞相互沟通对正常细胞和组织功能的要求 通过称为缝隙连接的特殊通道直接相互连接。我们的 长期以来，实验室一直对研究这些细胞的功能作用感兴趣 用于交换小信号和调节分子的通道 在细胞之间，也起到半通道的作用，向外释放分子 牢房。这些独特的大孔道是由一个巨大的 被称为连接蛋白(Cx)的蛋白质(在小鼠中n=20)。原因是我们做得不好 了解，许多细胞类型需要产生多个连接蛋白成员 从表达的连接蛋白的组合中构建缝隙连接通道。 这种现象在皮肤的表皮中最为明显，至少有7 连接蛋白(称为角质形成细胞连接蛋白)被表达。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。这项研究是 旨在确定角质形成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。我们的假设是角质形成细胞连接蛋白参与 经常与Cx43不同的贩运路线和监管集会活动 (一种主要的角质形成细胞连接蛋白)，导致形成不同的 正常的上皮细胞功能所需的通道。这些 研究尤其重要，因为新合成的连接蛋白可能是 靶向；a)质膜 没有作为半通道的缝隙连接，b)它们参与的缝隙连接复合体 在传统的缝隙连接细胞间通讯或c)线粒体内膜。我们的研究将会进行 在具有分化为角化细胞能力的大鼠角质形成细胞中 当表皮生长在液体/空气界面时。未标记的连接蛋白和 标记有荧光蛋白的连接蛋白将被使用，因为这允许 用于评估固定细胞和活细胞中的连接蛋白生命周期。我们 将使用药理学方法和荧光漂白策略来同时 确定多种连接蛋白的动态性质并解剖生命 每种连接蛋白的循环和组装特性。在某些情况下，我们预计 连接蛋白将在线粒体中占据一席之地 在角质形成细胞走向程序性细胞死亡的过程中对其进行调控。一旦我们 解析每个角质形成细胞连接蛋白的生命周期并确定其 在活体角质形成细胞和器官型表皮中的主要居留部位 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 不同连接蛋白组成的通道的定位和功能 在表皮更新方面。