Näthke I S, Hinck L, Swedlow J R, Papkoff J, Nelson W J
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, California 94305-5426.
J Cell Biol. 1994 Jun;125(6):1341-52. doi: 10.1083/jcb.125.6.1341.
The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well as the initiation and maintenance of structural and functional organization of cells and tissues. In the preceding study, we showed that the assembly of the cadherin/catenin complex is temporally regulated, and that novel combinations of catenin and cadherin complexes are formed in both Triton X-100-soluble and -insoluble fractions; we proposed a model in which pools of catenins are important in regulating assembly of E-cadherin/catenin and catenin complexes. Here, we sought to determine the spatial distributions of E-cadherin, alpha-catenin, beta-catenin, and plakoglobin, and whether different complexes of these proteins accumulate at steady state in polarized Madin-Darby canine kidney cells. Protein distributions were visualized by wide field, optical sectioning, and double immunofluorescence microscopy, followed by reconstruction of three-dimensional images. In cells that were extracted with Triton X-100 and then fixed (Triton X-100-insoluble fraction), more E-cadherin was concentrated at the apical junction relative to other areas of the lateral membrane. alpha-Catenin and beta-catenin colocalize with E-cadherin at the apical junctional complex. There is some overlap in the distribution of these proteins in the lateral membrane, but there are also areas where the distributions are distinct. Plakoglobin is excluded from the apical junctional complex, and its distribution in the lateral membrane is different from that of E-cadherin. Cells were also fixed and then permeabilized to reveal the total cellular pool of each protein (Triton X-100-soluble and -insoluble fractions). This analysis showed lateral membrane localization of alpha-catenin, beta-catenin, and plakoglobin, and it also revealed that they are distributed throughout the cell. Chemical cross-linking of proteins and analysis with specific antibodies confirmed the presence at steady state of E-cadherin/catenin complexes containing either beta-catenin or plakoglobin, and catenin complexes devoid of E-cadherin. Complexes containing E-cadherin/beta-catenin and E-cadherin/alpha-catenin are present in both the Triton X-100-soluble and -insoluble fractions, but E-cadherin/plakoglobin complexes are not detected in the Triton X-100-insoluble fraction. Taken together, these results show that different complexes of cadherin and catenins accumulate in fully polarized epithelial cells, and that they distribute to different sites. We suggest that cadherin/catenin and catenin complexes at different sites have specialized roles in establishing and maintaining the structural and functional organization of polarized epithelial cells.
钙黏蛋白/连环蛋白复合体在细胞黏附、信号转导以及细胞和组织的结构与功能组织的起始和维持中发挥着重要作用。在之前的研究中，我们表明钙黏蛋白/连环蛋白复合体的组装受到时间调控，并且在Triton X - 100可溶性和不溶性组分中形成了连环蛋白和钙黏蛋白复合体的新组合；我们提出了一个模型，其中连环蛋白池在调节E - 钙黏蛋白/连环蛋白和连环蛋白复合体的组装中很重要。在此，我们试图确定E - 钙黏蛋白、α - 连环蛋白、β - 连环蛋白和桥粒斑珠蛋白的空间分布，以及这些蛋白质的不同复合体在极化的Madin - Darby犬肾细胞中是否在稳态时积累。通过宽视野、光学切片和双重免疫荧光显微镜观察蛋白质分布，随后重建三维图像。在用Triton X - 100提取然后固定的细胞（Triton X - 100不溶性组分）中，相对于侧膜的其他区域，更多的E - 钙黏蛋白集中在顶端连接部位。α - 连环蛋白和β - 连环蛋白在顶端连接复合体处与E - 钙黏蛋白共定位。这些蛋白质在侧膜中的分布有一些重叠，但也有分布不同的区域。桥粒斑珠蛋白被排除在顶端连接复合体之外，并且其在侧膜中的分布与E - 钙黏蛋白不同。细胞也被固定然后通透以显示每种蛋白质的总细胞池（Triton X - 100可溶性和不溶性组分）。该分析显示α - 连环蛋白、β - 连环蛋白和桥粒斑珠蛋白在侧膜定位，并且还揭示它们分布在整个细胞中。蛋白质的化学交联和用特异性抗体进行的分析证实了在稳态时存在含有β - 连环蛋白或桥粒斑珠蛋白的E - 钙黏蛋白/连环蛋白复合体，以及不含E - 钙黏蛋白的连环蛋白复合体。含有E - 钙黏蛋白/β - 连环蛋白和E - 钙黏蛋白/α - 连环蛋白的复合体存在于Triton X - 100可溶性和不溶性组分中，但在Triton X - 100不溶性组分中未检测到E - 钙黏蛋白/桥粒斑珠蛋白复合体。综上所述，这些结果表明钙黏蛋白和连环蛋白的不同复合体在完全极化的上皮细胞中积累，并且它们分布到不同的位点。我们认为不同位点的钙黏蛋白/连环蛋白和连环蛋白复合体在建立和维持极化上皮细胞的结构与功能组织中具有特定作用。
