Salas P J, Rodriguez M L, Viciana A L, Vega-Salas D E, Hauri H P
Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida 33101, USA.
J Cell Biol. 1997 Apr 21;137(2):359-75. doi: 10.1083/jcb.137.2.359.
In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145-3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40-70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical-basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37 degrees C or in n-octyl-beta-D-glycoside at 4 degrees C (representative of GPI-anchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na+-K+ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.
在之前的一篇论文中（罗德里格斯，M.L.，M. 布里尼奥尼，以及 P.J.I. 萨拉斯。1994 年。《细胞科学杂志》107 卷：3145 - 3151 页），我们描述了在非刷状缘细胞中存在一种终末网样结构，它包含一种特异性定位于顶端的细胞角蛋白，推测为细胞角蛋白 19。在本研究中，我们证实了细胞角蛋白 19 的顶端分布，并将这一观察结果扩展到组织培养和体内的其他上皮细胞。在组织培养中，用两种靶向细胞角蛋白 19 mRNA 的不同 21 聚体硫代磷酸酯寡脱氧核苷酸持续处理亚汇合细胞株，使我们能够获得汇合的单层细胞，其中这种蛋白质部分（40 - 70%）且短暂减少。其他细胞骨架蛋白的表达未受干扰。细胞角蛋白 19 的这种下调导致：（a）微绒毛数量减少；（b）顶端（而非侧面或基部）丝状肌动蛋白紊乱以及顶端微管异常；（c）通过差异顶端 - 基底外侧生物素化测定，顶端膜蛋白耗竭或重新分布。事实上，在细胞角蛋白 19 水平较低的细胞中，一部分去污剂不溶性蛋白未在细胞表面表达。在 Triton X - 114 的去污剂相中纯化的顶端蛋白（通常是整合膜蛋白）以及在 37℃下用 Triton X - 100 或在 4℃下用正辛基 -β - D - 糖苷差异提取的蛋白（代表糖基磷脂酰肌醇锚定蛋白），似乎部分重新分布到基底外侧结构域。一种跨膜顶端蛋白，蔗糖酶异麦芽糖酶，在用反义寡核苷酸处理的细胞亚群中被发现极性错误，而 Na⁺ - K⁺ATP 酶的基底外侧极性未受影响。通过差异生物素化、亲和纯化和免疫印迹测定，在 A19 处理的 CACO - 2 单层细胞中，蔗糖酶异麦芽糖酶和碱性磷酸酶（一种糖基磷脂酰肌醇锚定蛋白）似乎都部分去极化。这些结果表明，中间丝的顶端亚膜细胞骨架在许多上皮细胞中表达，包括那些没有刷状缘的上皮细胞，尽管它可能并不普遍。此外，这些数据表明这种结构参与细胞质顶端区域和顶端膜的组织。
