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

1. 我们使用connexin 43-绿色荧光蛋白（Cx43-GFP）融合蛋白结合共聚焦激光扫描显微镜技术实时可视化活细胞缝隙连接。将大鼠Cx43基因与编码GFP的基因融合，并转染到人HeLa细胞中，产生稳定的组成表达Cx43-GFP的细胞系。在含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融合蛋白主要定位于接触细胞间质膜上的斑点、斑块或细胞系，而不含Cx43的GFP则在细胞质和细胞核中弥漫性表达。利用溴化乙啶染料显微注射转移法检测细胞间隙连接的通讯，发现Cx43-GFP融合蛋白形成了功能性的间隙连接。激光漂白后的延时显微镜显示，Cx43-GFP融合蛋白不仅在细胞膜上动态移动，而且在细胞质中动态移动。我们使用Cx43-GFP融合蛋白和荧光Ca2+指示剂（Fura Red）实时可视化间隙连接分布和细胞功能。用脂肪转染法将Cx43-EGFP载体转染培养的新生大鼠心肌细胞。同时可视化Cx43- egfp和细胞内Ca2+表明，Ca2+在表达Cx43- egfp的肌细胞和不表达Cx43- egfp的肌细胞之间是同步的，这表明Cx43- egfp不抑制内源性Cx43在心肌细胞中表达介导的间隙连接通讯。连接素- gfp和细胞功能的同时可视化为间隙连接的研究提供了一个有用的系统。利用显性阴性Cx43-GFP表达载体抑制间隙连接细胞间通讯我们通过定点诱变制备了显性阴性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（LAR20）时，通过野生型Cx43的间隙连接细胞间通讯以显性负向方式被抑制。这个向量为研究间隙连接提供了一个有用的系统。少

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