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Real-time and simulataneous analysis of gap junctional structure and function in living cells

活细胞间隙连接结构和功能的实时同步分析

基本信息

批准号：
10670214
负责人：
OYAMADA Masahito
金额：
$ 2.11万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670214/
关键词：
Intercellular communication Gap junctions Connexin Green fluorescent protein Confocal laser scanning microscopy Calcium imaging GFP マイクロインジェクション Hela細胞

项目摘要

1. Real-time visualization of gap junctions in living cells using connexin 43-GFP fusion proteinWe used connexin 43-green fluorescent protein (Cx43-GFP) fusion protein in combination with confocal laser scanning microscopy. The rat Cx43 gene was fused to the gene encoding GFP and transfected into human HeLa cells to generate stable lines constitutively expressing Cx43-GFP. Permanent cell lines expressing Cx43-GFP fusion protein and only GFP were obtained by culture in selection medium containing G418. Cx43-GFP fusion protein was localized as spots, plaques or lines mainly on plasma membranes between cells in contact, whereas GFP without Cx43 was expressed diffusely in the cytoplasm and nucleus. An assay of gap junctional intercellular communication using ethidium bromide dye microinjection transfer revealed that Cx43-GFP fusion protein formed functional gap junctions. Time-lapse microscopy after laser photobleaching showed that Cx43-GFP fusion protein dynamically moved not only on plas … More ma membranes but also in cytoplasm.2. Real-time visualization of gap junction distribution and cellular functionWe used Cx43-GFP fusion protein and a fluorescent Ca2+ indicator (Fura Red). Cx43-EGFP vector was transfected into cultured neonatal rat cardiomyocytes by lipofection. Simultaneous visualization of Cx43-EGFP and intracellular Ca2+ demonstrated that Ca2+ transients were synchronized between Cx43-EGFP-expressing myocytes and non-expressing myocytes, indicating that Cx43-EGFP did not inhibit gap junctional communication mediated by endogenous Cx43 expressed in the cardiomyocytes. The simultaneous visualization of connexin-GFP and cellular function provides a useful system for studies of gap junctions.3. Inhibition of gap junctional intercellular communication by use of dominant-negative Cx43-GFP expression vectorWe made a dominant-negative Cx43-green fluorescent protein (GFP) expression vector by sitedirected mutagenesis. When this vector was transfected into a liver epithelial cell line (LAR20) expressing wild-type Cx43, gap junctional intercellular communication via wild-type Cx43 was inhibited in dominant negative manners. This vector provides a useful system for studies of gap junctions. Less

1.利用Cx43-GFP融合蛋白实时观察活细胞间隙连接我们将Cx43-绿色荧光蛋白（Cx43-GFP）融合蛋白与共聚焦激光扫描显微镜相结合。将大鼠Cx43基因与编码GFP的基因融合，并转染到人HeLa细胞中，以产生组成型表达Cx43-GFP的稳定细胞系。通过在含有G418的选择培养基中培养获得表达Cx43-GFP融合蛋白和仅表达GFP的永久细胞系。Cx43-GFP融合蛋白主要以斑点、斑块或线条的形式定位于细胞间的质膜上，而不含Cx43的GFP则在细胞质和细胞核中弥散表达。用溴化乙锭染料显微注射转移的间隙连接细胞间通讯的测定显示，Cx43-GFP融合蛋白形成功能性间隙连接。激光光漂白后的延时显微镜显示，Cx43-GFP融合蛋白不仅在质粒上动态移动， ...更多信息 MA膜，但也在细胞质中。缝隙连接分布和细胞功能的实时可视化我们使用Cx43-GFP融合蛋白和荧光Ca 2+指示剂（Fura Red）。脂质体转染法将Cx43-EGFP载体转染培养的乳鼠心肌细胞。Cx43-EGFP和细胞内Ca 2+的同时可视化表明，Ca 2+瞬变在Cx43-EGFP表达的心肌细胞和非表达的心肌细胞之间是同步的，表明Cx43-EGFP不抑制心肌细胞中内源性Cx43表达介导的间隙连接通讯。连接蛋白-GFP和细胞功能的同时可视化为缝隙连接的研究提供了一个有用的系统.利用显性负性Cx43-GFP表达载体抑制细胞间隙连接通讯我们通过定点突变的方法构建了显性负性Cx43-绿色荧光蛋白（GFP）表达载体。当该载体转染到表达野生型Cx43的肝上皮细胞系（LAR 20）中时，通过野生型Cx43的间隙连接细胞间通讯以显性负性方式被抑制。该载体为缝隙连接的研究提供了一个有用的系统。少