THE APICAL SUBMEMBRANE CYTOSKELETON PARTICIPATES IN THE ORGANIZATION OF THE APICAL POLE IN EPITHELIAL-CELLS

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Sa las. 1994. J. Cell Sci. 107: 3145-3151), we described the existence of a terminal web-like structure in nonbrush border cells, which compris es a specifically apical cytokeratin, presumably cytokeratin 19. In th e present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue cul ture and in vivo. In tissue culture, subconfluent cell stocks under co ntinuous treatment with two different 21-mer phosphorothioate oligodeo xy 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 und isturbed. This downregulation of cytokeratin 19 resulted in (a) decrea se in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubu les; and (c) depletion or redistribution of apical membrane proteins a s determined by differential apical-basolateral biotinylation. In fact , a subset of detergent-insoluble proteins was not expressed on the ce ll surface in cells with lower levels of cytokeratin 19. Apical protei ns 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 (repres entative of GPI-anchored proteins), appeared partially redistributed t o the basolateral domain. A transmembrane apical protein, sucrase isom altase, 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 ph osphatase (a GPI-anchored protein) appeared partially depolarized in A 19 treated CACO-2 monolayers as determined by differential biotinylati on, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expres sed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate tha t this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.