Bacallao R, Antony C, Dotti C, Karsenti E, Stelzer E H, Simons K
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
J Cell Biol. 1989 Dec;109(6 Pt 1):2817-32. doi: 10.1083/jcb.109.6.2817.
Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4-nitrobenzo-2-oxa-1,3-diazole]) aminocaproyl sphingosine. 1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved greater than 10 microns upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical region of the cell and their plus ends toward the basal region. These findings show that there is an extensive remodeling of epithelial cytoarchitecture after formation of cell-cell contacts. Reorganization of the microtubule network results in functional polarization of the cytoplasm.
对小鼠胚胎中发育的滋养外胚层的研究表明，上皮形成过程中会发生广泛的细胞重塑。在本研究中，共聚焦免疫荧光显微镜用于检查终末分化上皮细胞系极化过程中细胞结构的三维变化。将Madin-Darby犬肾细胞低密度接种在可渗透滤膜支持物上。使用特异性识别紧密连接、黏附连接、微管、中心体和高尔基体复合体成分的抗体，研究极化细胞层形成过程中细胞结构的空间重塑。免疫荧光数据与通过跨上皮电阻和细胞表面反向交换测量的功能性紧密连接的建立相关，细胞表面用荧光脂质类似物N-(7-[4-硝基苯并-2-恶唑-1,3-二氮杂环])氨基己酰鞘氨醇的代谢物标记。接种后1天，单细胞有从包含中心体和高尔基体复合体的宽阔区域辐射出的微管。接种后2天，细胞生长至汇合，并在靠近基质处形成功能性紧密连接。中心粒已分裂，不再组织在细胞核上方和下方运行的微管。高尔基体复合体已扩散到细胞核周围。接种后第五天，达到最终极化状态。连接复合体已从其基部位置向上移动超过10微米。中心粒聚集在顶膜下方，高尔基体复合体形成位于细胞核上方顶区的带状卷曲结构。微管在柱状细胞的顶-基轴上组织成顶网和纵向微管束。纵向微管排列成其负端分布在细胞顶区，正端朝向基区。这些发现表明，细胞间接触形成后上皮细胞结构发生了广泛重塑。微管网络的重组导致细胞质功能极化。
