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The apical submembrane cytoskeleton participates in the organization of the apical pole in epithelial cells.

Salas PJ, Rodriguez ML, Viciana AL, Vega-Salas DE, Hauri HP - J. Cell Biol. (1997)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida 33101, USA.

ABSTRACT
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.

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Scanning electron microscopy of the apical surface of  MCF-10A (a and b) and CACO-2 (c and d) cells continuously  grown in random (a and c) or A19 (b and d) oligonucleotides.  Specimens were tilted 60° for photography. Bar, 1 μm.
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Figure 5: Scanning electron microscopy of the apical surface of MCF-10A (a and b) and CACO-2 (c and d) cells continuously grown in random (a and c) or A19 (b and d) oligonucleotides. Specimens were tilted 60° for photography. Bar, 1 μm.

Mentions: An intriguing observation in the immunoperoxidase experiments was the decrease in the number of microvilli in those cells with reduced levels of CK19 filaments (Fig. 3, c versus a, *). To further explore this phenomenon, MCF10A (Fig. 5, a and b) and CACO-2 cells (Fig. 5, c and d) continuously grown in random or A19 oligonucleotides were observed by scanning electron microscopy. Control MCF-10A cells displayed a relatively modest amount of short apical microvilli (Fig. 5 a), which was significantly decreased in nearly 30% of the cells in monolayers treated with A19 (Fig. 5 b). Control CACO-2 cells at 9 d of confluency showed a fully developed apical domain with abundant long microvilli (Fig. 5 c). In treated cultures, 30–40% of cells were totally depleted in microvilli (Fig. 5 d) and a similar proportion of cells had a decreased number of microvilli, as compared with the control.


The apical submembrane cytoskeleton participates in the organization of the apical pole in epithelial cells.

Salas PJ, Rodriguez ML, Viciana AL, Vega-Salas DE, Hauri HP - J. Cell Biol. (1997)

Scanning electron microscopy of the apical surface of  MCF-10A (a and b) and CACO-2 (c and d) cells continuously  grown in random (a and c) or A19 (b and d) oligonucleotides.  Specimens were tilted 60° for photography. Bar, 1 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139782&req=5

Figure 5: Scanning electron microscopy of the apical surface of MCF-10A (a and b) and CACO-2 (c and d) cells continuously grown in random (a and c) or A19 (b and d) oligonucleotides. Specimens were tilted 60° for photography. Bar, 1 μm.
Mentions: An intriguing observation in the immunoperoxidase experiments was the decrease in the number of microvilli in those cells with reduced levels of CK19 filaments (Fig. 3, c versus a, *). To further explore this phenomenon, MCF10A (Fig. 5, a and b) and CACO-2 cells (Fig. 5, c and d) continuously grown in random or A19 oligonucleotides were observed by scanning electron microscopy. Control MCF-10A cells displayed a relatively modest amount of short apical microvilli (Fig. 5 a), which was significantly decreased in nearly 30% of the cells in monolayers treated with A19 (Fig. 5 b). Control CACO-2 cells at 9 d of confluency showed a fully developed apical domain with abundant long microvilli (Fig. 5 c). In treated cultures, 30–40% of cells were totally depleted in microvilli (Fig. 5 d) and a similar proportion of cells had a decreased number of microvilli, as compared with the control.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida 33101, USA.

ABSTRACT
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.

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