Limits...
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.

Show MeSH
Distribution of CK19 in epithelial cells in vivo. Frozen  sections of fresh, unfixed human stomach (a–d) and rat small intestine (e and f) were processed for indirect immunofluorescence  with anti-CK19 mAb (A53-B/A2); (a and b) stomach, surface epithelium; (c and d) stomach, gland; (e and f) adult rat small intestine, epithelium of villi. Phase contrast images corresponding to  a, c, and e, are shown in b, d, and f. Nu indicates the position of  two nuclei taken as examples. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139782&req=5

Figure 12: Distribution of CK19 in epithelial cells in vivo. Frozen sections of fresh, unfixed human stomach (a–d) and rat small intestine (e and f) were processed for indirect immunofluorescence with anti-CK19 mAb (A53-B/A2); (a and b) stomach, surface epithelium; (c and d) stomach, gland; (e and f) adult rat small intestine, epithelium of villi. Phase contrast images corresponding to a, c, and e, are shown in b, d, and f. Nu indicates the position of two nuclei taken as examples. Bars, 10 μm.

Mentions: The results shown in the previous sections suggest that CK19 intermediate filaments play a role in the organization of the apical domain in tissue culture cells. To weigh its potential biological importance, we localized CK19, using specific mAbs and a polyclonal Ab, in a variety of epithelial tissues in vivo, in addition to the three epithelial cell lines (MDCK, CACO-2, and MCF-10, derived from mesoderm, endoderm and ectoderm, respectively). Special attention was placed on the subcellular distribution of CK19 in tissues in vivo. Although immunoperoxidase has been commonly used for these types of studies, we preferred immunofluorescence, with the understanding that it may provide a more precise subcellular localization of the CK19 epitopes. Three major morphological patterns of localization were observed: (a) continuous apical submembrane localization, with or without extensions toward the lateral domain, as previously observed in tissue culture cells (Fig. 12, a and c, surface and glandular epithelia of human stomach). (b) Broad distribution of CK19 in the apical cytoplasm was observed, for example, in the adult rat small intestine (Fig. 12 e). In this case, the signal was always supranuclear but almost negative under the apical domain itself, in which the terminal web was in turn positive for the broad spectrum anti-cytokeratin antibody (AE1/AE3; not shown). In addition, it must be noted that the enterocytes at the base of the crypts were negative and that the expression of CK19 correlated with the pathway of differentiation, with an increasing expression level as the cells displaced toward the opening of the crypts and the tip of the villi. (c) Some epithelia were found to be negative to CK19. A summary of these observations (including two additional reports by other groups) is presented in Table III. These data indicate that CK19 is apically polarized in a variety of epithelia, although not universal.


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)

Distribution of CK19 in epithelial cells in vivo. Frozen  sections of fresh, unfixed human stomach (a–d) and rat small intestine (e and f) were processed for indirect immunofluorescence  with anti-CK19 mAb (A53-B/A2); (a and b) stomach, surface epithelium; (c and d) stomach, gland; (e and f) adult rat small intestine, epithelium of villi. Phase contrast images corresponding to  a, c, and e, are shown in b, d, and f. Nu indicates the position of  two nuclei taken as examples. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 12: Distribution of CK19 in epithelial cells in vivo. Frozen sections of fresh, unfixed human stomach (a–d) and rat small intestine (e and f) were processed for indirect immunofluorescence with anti-CK19 mAb (A53-B/A2); (a and b) stomach, surface epithelium; (c and d) stomach, gland; (e and f) adult rat small intestine, epithelium of villi. Phase contrast images corresponding to a, c, and e, are shown in b, d, and f. Nu indicates the position of two nuclei taken as examples. Bars, 10 μm.
Mentions: The results shown in the previous sections suggest that CK19 intermediate filaments play a role in the organization of the apical domain in tissue culture cells. To weigh its potential biological importance, we localized CK19, using specific mAbs and a polyclonal Ab, in a variety of epithelial tissues in vivo, in addition to the three epithelial cell lines (MDCK, CACO-2, and MCF-10, derived from mesoderm, endoderm and ectoderm, respectively). Special attention was placed on the subcellular distribution of CK19 in tissues in vivo. Although immunoperoxidase has been commonly used for these types of studies, we preferred immunofluorescence, with the understanding that it may provide a more precise subcellular localization of the CK19 epitopes. Three major morphological patterns of localization were observed: (a) continuous apical submembrane localization, with or without extensions toward the lateral domain, as previously observed in tissue culture cells (Fig. 12, a and c, surface and glandular epithelia of human stomach). (b) Broad distribution of CK19 in the apical cytoplasm was observed, for example, in the adult rat small intestine (Fig. 12 e). In this case, the signal was always supranuclear but almost negative under the apical domain itself, in which the terminal web was in turn positive for the broad spectrum anti-cytokeratin antibody (AE1/AE3; not shown). In addition, it must be noted that the enterocytes at the base of the crypts were negative and that the expression of CK19 correlated with the pathway of differentiation, with an increasing expression level as the cells displaced toward the opening of the crypts and the tip of the villi. (c) Some epithelia were found to be negative to CK19. A summary of these observations (including two additional reports by other groups) is presented in Table III. These data indicate that CK19 is apically polarized in a variety of epithelia, although not universal.

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