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© The Rockefeller University Press, 0021-9525/1997//359 $5.00
The Journal of Cell Biology, Volume 137, Number 2, , 1997 359-375

Article

The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells



Pedro J.I. Salas*, Marcelo L. Rodriguez{ddagger}, Ana L. Viciana§, Dora E. Vega-Salas*, and Hans-Peter Hauri||

* Department of Cell Biology and Anatomy, and § Department of Pathology, University of Miami School of Medicine, Miami, Florida 33101; {ddagger} Department of Carcinogenesis, M.D. Anderson Cancer Center, University of Texas, Smithville, Texas 78957; and CH-4056 || Biozentrum, University of Basel, Department of Pharmacology, Basel CH-4056, Switzerland

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°C or in n-octyl-β-D-glycoside at 4°C (representative of GPIanchored 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.

1. Abbreviations used in this paper: CK, cytokeratin; F-actin, filamentous actin; IF, intermediate filament; TER, trans-epithelial electrical resistance.

The authors are indebted to Dr. W. James Nelson (Stanford University School of Medicine, Stanford, CA) for generously providing us with antiNa+–K+ATPase and anti-fodrin antibodies. We are also grateful to Dr. Rudolf Werner and Ms. Debbie Bradley, DNA Core Laboratory, Department of Biochemistry and Molecular Biology, for their assistance with the synthesis and purification of phosphorothioate and biotinylated deoxy oligonucleotides, to Dr. John N. Barret (Department of Physiology and Biophysics) for his advise in confocal microscopy, and to Dr. Kermit L. Carraway (University of Mami School of Medicine, Miami, FL) for critically reading the manuscript. It is a pleasure to thank Ms. JoAnne Anderson and Ms. Susan Decker for their proficient technical aid.

This paper is supported by grants 93GIA-949 from American Heart Association, Florida Affiliate, and K4HL03397A from National Institutes of Health.

Please address all correspondence to Pedro Salas, Department of Cell Biology and Anatomy, R-124, University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101. Tel.: (305) 243-6977; Fax: (305) 545-7166.


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