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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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Effect of A19 oligonucleotide on the polarity of sucrase isomaltase and alkaline phosphatase in CACO-2 cells. Confluent monolayers were grown on polycarbonate filters in the  presence of random (lanes A and B) or A19 (lanes C and D) oligonucleotides as described above. The cells were biotinylated  from the apical (lanes A and C) or basolateral sides (lanes B and  D) and consecutively extracted in Triton X-114 at 4°C and with  Triton X-100 at 37°C. The Triton X-114 detergent phase and the  Triton X-100 supernatant were pooled and affinity purified with  streptavidin–agarose. The eluates or the affinity purification step  were then analyzed by immunoblot with antibodies against either  sucrase isomaltase or alkaline phosphatase and a chemiluminescence detection system. The average OD measures obtained  from unfiltered digitized images after subracting background  from the average pixel value over the band of each lane were as  follows (scale 0-255): (Sucrase A) 58; (B) 14; (C) 55; (D) 29; (Alk.  Phosphatase, A) 74; (B) 1; (C) 30; (D) 16. Weighted OD measures (average pixel value × number of pixels) were as follows  (in thousands): (Sucrase, A) 141; (B) 10; (C) 169; (D) 35; (Alk.  Phosphatase, A) 45; (B) 0.6; (C) 36; (D) 5.
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Figure 11: Effect of A19 oligonucleotide on the polarity of sucrase isomaltase and alkaline phosphatase in CACO-2 cells. Confluent monolayers were grown on polycarbonate filters in the presence of random (lanes A and B) or A19 (lanes C and D) oligonucleotides as described above. The cells were biotinylated from the apical (lanes A and C) or basolateral sides (lanes B and D) and consecutively extracted in Triton X-114 at 4°C and with Triton X-100 at 37°C. The Triton X-114 detergent phase and the Triton X-100 supernatant were pooled and affinity purified with streptavidin–agarose. The eluates or the affinity purification step were then analyzed by immunoblot with antibodies against either sucrase isomaltase or alkaline phosphatase and a chemiluminescence detection system. The average OD measures obtained from unfiltered digitized images after subracting background from the average pixel value over the band of each lane were as follows (scale 0-255): (Sucrase A) 58; (B) 14; (C) 55; (D) 29; (Alk. Phosphatase, A) 74; (B) 1; (C) 30; (D) 16. Weighted OD measures (average pixel value × number of pixels) were as follows (in thousands): (Sucrase, A) 141; (B) 10; (C) 169; (D) 35; (Alk. Phosphatase, A) 45; (B) 0.6; (C) 36; (D) 5.

Mentions: Our previous observation of an apically distributed cytokeratin displaying significant peptide homologies with cytokeratin 19 was further confirmed by cross-reaction of antibodies. Most mAbs against CK19 did not cross-react with canine cells, including the mAb against nonhuman CK19 (MAB1675). One of them (K4.62) however, did recognize the same protein that our polyclonal antibody localized in the apical domain of MDCK cells (Rodriguez et al., 1994). Conversely, our polyclonal antibody against the apical cytokeratin in MDCK cells recognized the same band in immunoblots from human cells as all of the anti-CK19 mAbs (Table I). It consistently showed the same apical subcellular distribution (see Table III) in immunofluorescence as the anti-CK19 mAbs in tissue culture cell lines and epithelial tissues from biopsies (RCK108 and A53-B/A2, Fig. 2, g and h, and see Fig. 11). On the basis of these experiments we confirmed that human CK19 also exhibits an apical distribution in various epithelia. To assess whether these apical CK19 intermediate filaments play any role in the organization of the apical region, we attempted its knockout using a phosphorothioate deoxyoligonucleotide with the antisense sequence of the first 21 bases in the open reading frame of human CK19 mRNA (Eckert, 1988), hereafter referred to as A19 (Fig. 1). As a control, we used another 21-mer phosphorothioate deoxyoligonucleotide, with the same bases but in a randomized sequence (Fig. 1, random). In addition, another 21-mer antisense oligonucleotide, complementary to the last 11 bases in the 5′ UTR and the first 10 of the ORF of CK19 mRNA (A19/2) was also used in some experiments to further confirm the specificity of the effect and controlled with the corresponding randomized sequence oligonucleotide (Fig. 1, random/2). All the sequences were searched in the GenBank database. The 32 bases in the CK19 message (to which A19 and A19/2 were antisense sequences) showed high homologies with CK19 from other species but not with other known human mRNAs. The only other significant homologies found in vertebrates were proteins expressed in hippocampal neurons in rat (85% identity in 20 bases; these sequence data are available from GenBank/EMBL/DDBJ under accession no. L26525) and in T-cells in mice (89.5% identity in 19 bases; under accession no. M16122). Therefore, CK19 is likely to be the only protein targeted by A19 or A19/2 antisense oligonucleotides. In the case of the random oligonucleotides, no significant reverse/complemented homologies were found in higher eukaryote sequences. For all the experiments, the random oligonucleotides were synthesized at the same time with the same reagents and purified in parallel with the corresponding antisense oligonucleotides for CK19.


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)

Effect of A19 oligonucleotide on the polarity of sucrase isomaltase and alkaline phosphatase in CACO-2 cells. Confluent monolayers were grown on polycarbonate filters in the  presence of random (lanes A and B) or A19 (lanes C and D) oligonucleotides as described above. The cells were biotinylated  from the apical (lanes A and C) or basolateral sides (lanes B and  D) and consecutively extracted in Triton X-114 at 4°C and with  Triton X-100 at 37°C. The Triton X-114 detergent phase and the  Triton X-100 supernatant were pooled and affinity purified with  streptavidin–agarose. The eluates or the affinity purification step  were then analyzed by immunoblot with antibodies against either  sucrase isomaltase or alkaline phosphatase and a chemiluminescence detection system. The average OD measures obtained  from unfiltered digitized images after subracting background  from the average pixel value over the band of each lane were as  follows (scale 0-255): (Sucrase A) 58; (B) 14; (C) 55; (D) 29; (Alk.  Phosphatase, A) 74; (B) 1; (C) 30; (D) 16. Weighted OD measures (average pixel value × number of pixels) were as follows  (in thousands): (Sucrase, A) 141; (B) 10; (C) 169; (D) 35; (Alk.  Phosphatase, A) 45; (B) 0.6; (C) 36; (D) 5.
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Figure 11: Effect of A19 oligonucleotide on the polarity of sucrase isomaltase and alkaline phosphatase in CACO-2 cells. Confluent monolayers were grown on polycarbonate filters in the presence of random (lanes A and B) or A19 (lanes C and D) oligonucleotides as described above. The cells were biotinylated from the apical (lanes A and C) or basolateral sides (lanes B and D) and consecutively extracted in Triton X-114 at 4°C and with Triton X-100 at 37°C. The Triton X-114 detergent phase and the Triton X-100 supernatant were pooled and affinity purified with streptavidin–agarose. The eluates or the affinity purification step were then analyzed by immunoblot with antibodies against either sucrase isomaltase or alkaline phosphatase and a chemiluminescence detection system. The average OD measures obtained from unfiltered digitized images after subracting background from the average pixel value over the band of each lane were as follows (scale 0-255): (Sucrase A) 58; (B) 14; (C) 55; (D) 29; (Alk. Phosphatase, A) 74; (B) 1; (C) 30; (D) 16. Weighted OD measures (average pixel value × number of pixels) were as follows (in thousands): (Sucrase, A) 141; (B) 10; (C) 169; (D) 35; (Alk. Phosphatase, A) 45; (B) 0.6; (C) 36; (D) 5.
Mentions: Our previous observation of an apically distributed cytokeratin displaying significant peptide homologies with cytokeratin 19 was further confirmed by cross-reaction of antibodies. Most mAbs against CK19 did not cross-react with canine cells, including the mAb against nonhuman CK19 (MAB1675). One of them (K4.62) however, did recognize the same protein that our polyclonal antibody localized in the apical domain of MDCK cells (Rodriguez et al., 1994). Conversely, our polyclonal antibody against the apical cytokeratin in MDCK cells recognized the same band in immunoblots from human cells as all of the anti-CK19 mAbs (Table I). It consistently showed the same apical subcellular distribution (see Table III) in immunofluorescence as the anti-CK19 mAbs in tissue culture cell lines and epithelial tissues from biopsies (RCK108 and A53-B/A2, Fig. 2, g and h, and see Fig. 11). On the basis of these experiments we confirmed that human CK19 also exhibits an apical distribution in various epithelia. To assess whether these apical CK19 intermediate filaments play any role in the organization of the apical region, we attempted its knockout using a phosphorothioate deoxyoligonucleotide with the antisense sequence of the first 21 bases in the open reading frame of human CK19 mRNA (Eckert, 1988), hereafter referred to as A19 (Fig. 1). As a control, we used another 21-mer phosphorothioate deoxyoligonucleotide, with the same bases but in a randomized sequence (Fig. 1, random). In addition, another 21-mer antisense oligonucleotide, complementary to the last 11 bases in the 5′ UTR and the first 10 of the ORF of CK19 mRNA (A19/2) was also used in some experiments to further confirm the specificity of the effect and controlled with the corresponding randomized sequence oligonucleotide (Fig. 1, random/2). All the sequences were searched in the GenBank database. The 32 bases in the CK19 message (to which A19 and A19/2 were antisense sequences) showed high homologies with CK19 from other species but not with other known human mRNAs. The only other significant homologies found in vertebrates were proteins expressed in hippocampal neurons in rat (85% identity in 20 bases; these sequence data are available from GenBank/EMBL/DDBJ under accession no. L26525) and in T-cells in mice (89.5% identity in 19 bases; under accession no. M16122). Therefore, CK19 is likely to be the only protein targeted by A19 or A19/2 antisense oligonucleotides. In the case of the random oligonucleotides, no significant reverse/complemented homologies were found in higher eukaryote sequences. For all the experiments, the random oligonucleotides were synthesized at the same time with the same reagents and purified in parallel with the corresponding antisense oligonucleotides for CK19.

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