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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 antisense  A19 oligonucleotide on the  apical distribution of apical  tubulin in CACO-2 cells. The  cells were continuously grown  in random (a and c; control)  or antisense A19 (b and d)  oligonucleotides on glass coverslips for 9 d, saponin permeabilized, and fixed. The  tight junction component  ZO-1 (insets) and tubulin  were localized by indirect immunofluorescence using specific second antibodies coupled to fluorescein and Texas  red, respectively. Laser confocal optical sections were  taken simultaneously in the  green (ZO-1; insets) and red  at two different levels: through  the apical cytoplasm at the  deep side of the ZO-1 signal  (a, b) and ∼1 μm above the  basal membrane (c, d). The  pairs a–c and b–d correspond  respectively to the same fields  at different focal planes. Arrowheads point at tubulin  clusters. Bars, 10 μm (equivalent to 30.4 μm for the insets).
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Figure 8: Effect of antisense A19 oligonucleotide on the apical distribution of apical tubulin in CACO-2 cells. The cells were continuously grown in random (a and c; control) or antisense A19 (b and d) oligonucleotides on glass coverslips for 9 d, saponin permeabilized, and fixed. The tight junction component ZO-1 (insets) and tubulin were localized by indirect immunofluorescence using specific second antibodies coupled to fluorescein and Texas red, respectively. Laser confocal optical sections were taken simultaneously in the green (ZO-1; insets) and red at two different levels: through the apical cytoplasm at the deep side of the ZO-1 signal (a, b) and ∼1 μm above the basal membrane (c, d). The pairs a–c and b–d correspond respectively to the same fields at different focal planes. Arrowheads point at tubulin clusters. Bars, 10 μm (equivalent to 30.4 μm for the insets).

Mentions: The distribution of microtubules was slightly different in CACO-2 cells. These cells usually display a longer apical– basal axis than the MCF-10A cells. Because the nucleus is located near the basal region, they show a thicker apical cytoplasm. To demarcate regions within the apical cytoplasm we colocalized ZO-1, a tight-junction marker (Stevenson et al., 1986) together with tubulin. Confocal optical sections were collected in the green channel for ZO-1 (Fig. 8, insets) and in the red channel for tubulin (Texas red; right hand side of each pair in Fig. 8, a–d). As in Fig. 7, the cells were briefly permeabilized before fixation to remove the signal from nonpolymerized tubulin. In control monolayers (treated with random oligonucleotide), microtubules were practically excluded from the apical-most region of the cytoplasm, and a thick (3–4 μm) network of microtubules was observed (Fig. 8 a). This network extended from the level of the tight junctions to the cytoplasm immediately above the nucleus. At the nuclear level, the microtubules were mostly oriented in the apical–basal axis. It must be noted that these apical–basal microbutules appear in confocal sections as small dots, giving an impression of poor preservation. Standard epifluorescence of these preparations showed images comparable with those in Fig. 7. Finally, the basal cytoplasm showed another network of microtubules, thinner than the apical one (Fig. 8 c). These results are coincidental with the observations of Gilbert et al. (1991) in these cells. The downregulation of CK19 with A19 had a striking effect on the apical microtubules. Microtubules were, again, mostly excluded from the apical-most cytoplasm as in the controls, but the apical network showed images of thick spherically shaped (caliper diameters 0.3– 1.2 μm) clusters of tubulin (Fig. 8 b, arrowheads). These images were not continuous in deeper sections, indicating that they were not thick bundles perpendicular to the plane of the monolayer. Some of these structures were also observed at the nuclear level, intercalated with normal microtubules. In the basal cytoplasm, the fine basal network of microtubules was indistinguishable from the control images (Fig. 8 d).


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 antisense  A19 oligonucleotide on the  apical distribution of apical  tubulin in CACO-2 cells. The  cells were continuously grown  in random (a and c; control)  or antisense A19 (b and d)  oligonucleotides on glass coverslips for 9 d, saponin permeabilized, and fixed. The  tight junction component  ZO-1 (insets) and tubulin  were localized by indirect immunofluorescence using specific second antibodies coupled to fluorescein and Texas  red, respectively. Laser confocal optical sections were  taken simultaneously in the  green (ZO-1; insets) and red  at two different levels: through  the apical cytoplasm at the  deep side of the ZO-1 signal  (a, b) and ∼1 μm above the  basal membrane (c, d). The  pairs a–c and b–d correspond  respectively to the same fields  at different focal planes. Arrowheads point at tubulin  clusters. Bars, 10 μm (equivalent to 30.4 μm for the insets).
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Related In: Results  -  Collection

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Figure 8: Effect of antisense A19 oligonucleotide on the apical distribution of apical tubulin in CACO-2 cells. The cells were continuously grown in random (a and c; control) or antisense A19 (b and d) oligonucleotides on glass coverslips for 9 d, saponin permeabilized, and fixed. The tight junction component ZO-1 (insets) and tubulin were localized by indirect immunofluorescence using specific second antibodies coupled to fluorescein and Texas red, respectively. Laser confocal optical sections were taken simultaneously in the green (ZO-1; insets) and red at two different levels: through the apical cytoplasm at the deep side of the ZO-1 signal (a, b) and ∼1 μm above the basal membrane (c, d). The pairs a–c and b–d correspond respectively to the same fields at different focal planes. Arrowheads point at tubulin clusters. Bars, 10 μm (equivalent to 30.4 μm for the insets).
Mentions: The distribution of microtubules was slightly different in CACO-2 cells. These cells usually display a longer apical– basal axis than the MCF-10A cells. Because the nucleus is located near the basal region, they show a thicker apical cytoplasm. To demarcate regions within the apical cytoplasm we colocalized ZO-1, a tight-junction marker (Stevenson et al., 1986) together with tubulin. Confocal optical sections were collected in the green channel for ZO-1 (Fig. 8, insets) and in the red channel for tubulin (Texas red; right hand side of each pair in Fig. 8, a–d). As in Fig. 7, the cells were briefly permeabilized before fixation to remove the signal from nonpolymerized tubulin. In control monolayers (treated with random oligonucleotide), microtubules were practically excluded from the apical-most region of the cytoplasm, and a thick (3–4 μm) network of microtubules was observed (Fig. 8 a). This network extended from the level of the tight junctions to the cytoplasm immediately above the nucleus. At the nuclear level, the microtubules were mostly oriented in the apical–basal axis. It must be noted that these apical–basal microbutules appear in confocal sections as small dots, giving an impression of poor preservation. Standard epifluorescence of these preparations showed images comparable with those in Fig. 7. Finally, the basal cytoplasm showed another network of microtubules, thinner than the apical one (Fig. 8 c). These results are coincidental with the observations of Gilbert et al. (1991) in these cells. The downregulation of CK19 with A19 had a striking effect on the apical microtubules. Microtubules were, again, mostly excluded from the apical-most cytoplasm as in the controls, but the apical network showed images of thick spherically shaped (caliper diameters 0.3– 1.2 μm) clusters of tubulin (Fig. 8 b, arrowheads). These images were not continuous in deeper sections, indicating that they were not thick bundles perpendicular to the plane of the monolayer. Some of these structures were also observed at the nuclear level, intercalated with normal microtubules. In the basal cytoplasm, the fine basal network of microtubules was indistinguishable from the control images (Fig. 8 d).

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