Peterson M D, Mooseker M S
Department of Cell Biology, Yale University, New Haven, CT 06511-8112.
J Cell Sci. 1993 Jun;105 ( Pt 2):445-60. doi: 10.1242/jcs.105.2.445.
Intestinal epithelial cells assemble and maintain a polarized, highly organized membrane-cytoskeleton array, the brush border. We describe an in vitro, cell contact-induced brush border assembly model using the Caco-2BBe clones. Subconfluent cells were 'depolarized' by brief passage through suspension culture in the presence of cytochalasin D and re-plated on filters at high density in low-Ca2+ medium. Upon return to regular medium, these small, rounded cells with bleb-like protrusions formed, over the course of 19 days, a polarized monolayer of tall, columnar cells with a well-defined brush border. Ultrastructural changes were documented by both transmission and scanning electron microscopy. The earliest events of microvillar assembly coincided with a short period of cell aggregation. Intercellular cysts were occasionally observed within these aggregates, and junction formation between cells which had no contact with the filter was also observed. Monolayer formation was completed within 48 hours, and cell height steadily increased approximately 3.5-fold over 19 days. Concurrent with monolayer formation and the increase in cell height, sparse microvilli with a few actin core filaments gradually became more dense and better organized. By the third day, the actin core bundles had begun to extend into the subjacent cytoplasm, while terminal web assembly was underway at five days. The mature morphology of the brush border was first observed at nine days, although cell height and microvillar density continued to increase during the subsequent ten days. Microvillar density rose approximately nine-fold throughout brush border assembly in the Caco-2BBe cells. With the exception of the formation of cellular aggregates at the onset of the time course, this sequence of morphological changes is comparable to that observed during brush border assembly in embryonic intestinal epithelial cells. The Caco-2BBe assembly model provides a useful system in which to investigate various molecular aspects of brush border assembly.
肠上皮细胞组装并维持一种极化的、高度有序的膜 - 细胞骨架阵列，即刷状缘。我们描述了一种使用Caco - 2BBe克隆的体外细胞接触诱导刷状缘组装模型。亚汇合细胞在细胞松弛素D存在下通过悬浮培养短暂传代而“去极化”，然后在低钙培养基中高密度重新接种到滤膜上。回到常规培养基后，这些带有泡状突起的小圆形细胞在19天的过程中形成了一层极化的单层高柱状细胞，具有明确的刷状缘。通过透射电子显微镜和扫描电子显微镜记录了超微结构变化。微绒毛组装的最早事件与细胞聚集的短暂时期相吻合。在这些聚集体中偶尔观察到细胞间囊肿，并且还观察到与滤膜无接触的细胞之间的连接形成。单层形成在48小时内完成，细胞高度在19天内稳步增加约3.5倍。与单层形成和细胞高度增加同时发生的是，带有一些肌动蛋白核心丝的稀疏微绒毛逐渐变得更加密集且组织更好。到第三天，肌动蛋白核心束开始延伸到下方的细胞质中，而终末网组装在五天时开始。刷状缘的成熟形态在九天时首次观察到，尽管在随后的十天中细胞高度和微绒毛密度继续增加。在Caco - 2BBe细胞的刷状缘组装过程中微绒毛密度增加了约九倍。除了在时间进程开始时形成细胞聚集体外，这种形态变化序列与胚胎肠上皮细胞刷状缘组装过程中观察到的情况相当。Caco - 2BBe组装模型提供了一个有用的系统，可用于研究刷状缘组装的各种分子方面。
