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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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Immunoelectron  microscopy localization of  CK19 in MCF-10A cells. The  cells were continuously grown  in either random (a, b, and d)  or A19 (c and e) oligonucleotides on Transwell™ filters,  fixed, and processed for indirect immunoperoxidase with  anti-CK19 mAb (K4.62). (a  and c) Apical cytoplasm; (d  and e) basal cytoplasm; (b)  lateral domain. The black  arrowheads point at peroxidase positive intermediate filaments. (D) White arrowheads  show desmosomes; Nu, nucleus; *, microvillus. Bar: (a  and c–e) 0.52 μm; (b) 0.35 μm.
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Figure 3: Immunoelectron microscopy localization of CK19 in MCF-10A cells. The cells were continuously grown in either random (a, b, and d) or A19 (c and e) oligonucleotides on Transwell™ filters, fixed, and processed for indirect immunoperoxidase with anti-CK19 mAb (K4.62). (a and c) Apical cytoplasm; (d and e) basal cytoplasm; (b) lateral domain. The black arrowheads point at peroxidase positive intermediate filaments. (D) White arrowheads show desmosomes; Nu, nucleus; *, microvillus. Bar: (a and c–e) 0.52 μm; (b) 0.35 μm.

Mentions: Because the fluorescence label was dim in MCF-10A cells, we repeated these experiments using immunoperoxidase at the EM level. Control monolayers incubated in random oligonucleotide showed bundles of CK19 filaments in the apical cytoplasm (Fig. 3 a, arrowheads), at variable distances from the apical membrane. The minimum distance from these filaments to the apical membrane was ∼50 nm and the maximum up to 3 μm (sections from 22 cells). In a few cases, some filaments were observed around and below the nucleus but never close to the basal membrane. These filaments were never observed inside microvilli. In some cases, CK19 positive filaments were viewed along the lateral membrane (Fig. 3 b, arrowheads) in close vicinity to the desmosomes. The majority of the sections of the lateral domain, though, were negative for CK19. The basal cytoplasm on the other hand was always negative (Fig. 3 d). Monolayers incubated in A19 displayed nearly 50% of the apical cytoplasm sections without or with very few filaments (Fig. 3 c). In these cases, the filaments observed were found farther away from the apical membrane, with a minimum recorded distance of 320 nm (sections from 18 cells), and more scattered throughout the cytoplasm (Fig. 3, c and e).


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)

Immunoelectron  microscopy localization of  CK19 in MCF-10A cells. The  cells were continuously grown  in either random (a, b, and d)  or A19 (c and e) oligonucleotides on Transwell™ filters,  fixed, and processed for indirect immunoperoxidase with  anti-CK19 mAb (K4.62). (a  and c) Apical cytoplasm; (d  and e) basal cytoplasm; (b)  lateral domain. The black  arrowheads point at peroxidase positive intermediate filaments. (D) White arrowheads  show desmosomes; Nu, nucleus; *, microvillus. Bar: (a  and c–e) 0.52 μm; (b) 0.35 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139782&req=5

Figure 3: Immunoelectron microscopy localization of CK19 in MCF-10A cells. The cells were continuously grown in either random (a, b, and d) or A19 (c and e) oligonucleotides on Transwell™ filters, fixed, and processed for indirect immunoperoxidase with anti-CK19 mAb (K4.62). (a and c) Apical cytoplasm; (d and e) basal cytoplasm; (b) lateral domain. The black arrowheads point at peroxidase positive intermediate filaments. (D) White arrowheads show desmosomes; Nu, nucleus; *, microvillus. Bar: (a and c–e) 0.52 μm; (b) 0.35 μm.
Mentions: Because the fluorescence label was dim in MCF-10A cells, we repeated these experiments using immunoperoxidase at the EM level. Control monolayers incubated in random oligonucleotide showed bundles of CK19 filaments in the apical cytoplasm (Fig. 3 a, arrowheads), at variable distances from the apical membrane. The minimum distance from these filaments to the apical membrane was ∼50 nm and the maximum up to 3 μm (sections from 22 cells). In a few cases, some filaments were observed around and below the nucleus but never close to the basal membrane. These filaments were never observed inside microvilli. In some cases, CK19 positive filaments were viewed along the lateral membrane (Fig. 3 b, arrowheads) in close vicinity to the desmosomes. The majority of the sections of the lateral domain, though, were negative for CK19. The basal cytoplasm on the other hand was always negative (Fig. 3 d). Monolayers incubated in A19 displayed nearly 50% of the apical cytoplasm sections without or with very few filaments (Fig. 3 c). In these cases, the filaments observed were found farther away from the apical membrane, with a minimum recorded distance of 320 nm (sections from 18 cells), and more scattered throughout the cytoplasm (Fig. 3, c and e).

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.

Show MeSH