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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  polarity of plasma membrane  proteins in CACO-2 cells  C2BBe1. The cells were continuously grown in random  (lanes A, B, E, F, I, and J;  control) or antisense A19  (lanes C, D, G, H, K, and L)  oligonucleotides. For these  experiments, the cells were  plated on 24-mm TranswellTM  filters and cultured at confluency for 9 d. The monolayers  were biotinylated from either  the apical side (lanes A, C, E,  G, I, K; Ap) or from the basolateral side (lanes B, D, F,  H, J, L; Bl). Then, the filters  were extracted in ice-cold  PBS-EDTA supplemented  with 2% Triton X-114. The  supernatant of this extraction  was warmed to 30°C for 3 min,  and the detergent phase was  acetone precipitated and run  in SDS-PAGE (lanes I–L;  TX-114). The pellets from the  TX-114 extraction were then  resuspended in PBS-EDTA,  1% Triton X-100 by sonication, and warmed up to 37°C  for 15 min. The supernatants  (lanes E–H; TX-100) and pellets (lanes A–D; Pellet) of this  second extraction were also  run in SDS-PAGE. In all cases the total amount of protein was measured to ensure that all lanes for a given extraction procedure were  seeded with the same amounts of cellular material. All the lanes were blotted onto nitrocellulose sheets and probed with streptavidin–peroxidase and a chemiluminescence reaction. The small arrows between K and L point at apical bands now appearing also in the basolateral labeled set of proteins. The arrowheads indicate the position of molecular weight standards: (lanes A–D) 193, 112, 86, 70, 57, and 36  kD; (lanes E–L) 205, 116, 66, 45, and 29 kD. All the blots are from the same experiment, although, for technical reasons, they were run  in separate gels with two different sets of molecular weight standards. Biotinylation control: CACO-2 C2BBe monolayers were grown  on filters, incubated in A19, and biotinylated as described above. The cells were extensively washed, fixed in PFA, and processed with  fluorescein-coupled streptavidin from both sides of the filter. Optical confocal sections were taken at the transnuclear plane (a) or at the  apical membrane plane (b). Bars, 10 μm.
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Figure 9: Effect of antisense A19 oligonucleotide on the polarity of plasma membrane proteins in CACO-2 cells C2BBe1. The cells were continuously grown in random (lanes A, B, E, F, I, and J; control) or antisense A19 (lanes C, D, G, H, K, and L) oligonucleotides. For these experiments, the cells were plated on 24-mm TranswellTM filters and cultured at confluency for 9 d. The monolayers were biotinylated from either the apical side (lanes A, C, E, G, I, K; Ap) or from the basolateral side (lanes B, D, F, H, J, L; Bl). Then, the filters were extracted in ice-cold PBS-EDTA supplemented with 2% Triton X-114. The supernatant of this extraction was warmed to 30°C for 3 min, and the detergent phase was acetone precipitated and run in SDS-PAGE (lanes I–L; TX-114). The pellets from the TX-114 extraction were then resuspended in PBS-EDTA, 1% Triton X-100 by sonication, and warmed up to 37°C for 15 min. The supernatants (lanes E–H; TX-100) and pellets (lanes A–D; Pellet) of this second extraction were also run in SDS-PAGE. In all cases the total amount of protein was measured to ensure that all lanes for a given extraction procedure were seeded with the same amounts of cellular material. All the lanes were blotted onto nitrocellulose sheets and probed with streptavidin–peroxidase and a chemiluminescence reaction. The small arrows between K and L point at apical bands now appearing also in the basolateral labeled set of proteins. The arrowheads indicate the position of molecular weight standards: (lanes A–D) 193, 112, 86, 70, 57, and 36 kD; (lanes E–L) 205, 116, 66, 45, and 29 kD. All the blots are from the same experiment, although, for technical reasons, they were run in separate gels with two different sets of molecular weight standards. Biotinylation control: CACO-2 C2BBe monolayers were grown on filters, incubated in A19, and biotinylated as described above. The cells were extensively washed, fixed in PFA, and processed with fluorescein-coupled streptavidin from both sides of the filter. Optical confocal sections were taken at the transnuclear plane (a) or at the apical membrane plane (b). Bars, 10 μm.

Mentions: The cells were extracted at 0°C in Triton X-114, and the proteins were acetone precipitated from the 30°C detergent phase of the supernatant (Fig. 9, lanes I–L). These bands generally correspond to integral membrane proteins (Bordier, 1981). The pellets from the previous extraction were further extracted in Triton X-100 at 37°C. The proteins insoluble at 0–4°C but solubilized at a higher temperature typically correspond to (although perhaps are not exclusively) GPI-anchored membrane proteins (Brown and Rose, 1992; Fig. 9, lanes E–H). The results with this type of extraction were very similar to those obtained extracting the monolayers with 60 mM n-octyl-β-d-glycoside at 4°C after a Triton X-100 extraction at 0°C, another procedure to selectively extract GPI-anchored proteins (Hooper and Turner, 1988; Brown and Rose, 1992; and results not shown). Finally, the remaining membrane proteins in the pellet after both extractions are more likely to be truly associated with the submembrane cytoskeleton (Salas et al., 1988; Fig. 9, lanes A–D). Among the latter, the polarization was variable in control cells: at least five bands were distributed to both apical and basolateral domains, seven bands appeared mostly on the apical side, and 15–17 bands on the basolateral (Fig. 9, lanes A and B). A19 treated cells showed no detectable variations in the basolateral cytoskeletally associated membrane proteins (Fig. 9 D). The apical domain, on the other hand, displayed only three bands with levels of expression similar to the control (220, 194, and 72 kD). Two bands showed decreased levels (118 and 60 kD), while the rest were almost undetectable (Fig. 9 C). Coincidentally, the bands that were no longer expressed on the apical domain of A19 treated cells are in the same molecular weight range (40–86 kD) as those apical membrane proteins attached to CK19 multiprotein complexes in MDCK cells (Rodriguez et al., 1994). The bands extracted by Triton X-100 at 37°C after a previous extraction at 0°C showed a completely different pattern. As expected for GPI-anchored proteins (Lisanti et al., 1988), a vast majority of them localized to the apical domain in control cells (Fig. 9 E). In antisense treated cells, conversely, a number of bands appeared in the basolateral membrane (Fig. 9 H). Finally, the proteins extracted and purified in the detergent phase of Triton X-114 (integral membrane proteins; Bordier, 1981) showed a more complex pattern, with a number of bands polarized in either the apical or basolateral domains and at least four bands with the same molecular weights, presumably the same nonpolarized proteins, in both domains in control cells (Fig. 9, lanes I and J). Cells continuously grown in the presence of A19, however, showed five of the apical proteins redistributed to basolateral set (Fig. 9, marked by thin arrows between lanes K and L). Some proteins that remained nearly unchanged by the treatment with A19 have been marked with *. These examples, present in all lanes, rule out the possibility of variations in the efficiency of biotinylation.


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  polarity of plasma membrane  proteins in CACO-2 cells  C2BBe1. The cells were continuously grown in random  (lanes A, B, E, F, I, and J;  control) or antisense A19  (lanes C, D, G, H, K, and L)  oligonucleotides. For these  experiments, the cells were  plated on 24-mm TranswellTM  filters and cultured at confluency for 9 d. The monolayers  were biotinylated from either  the apical side (lanes A, C, E,  G, I, K; Ap) or from the basolateral side (lanes B, D, F,  H, J, L; Bl). Then, the filters  were extracted in ice-cold  PBS-EDTA supplemented  with 2% Triton X-114. The  supernatant of this extraction  was warmed to 30°C for 3 min,  and the detergent phase was  acetone precipitated and run  in SDS-PAGE (lanes I–L;  TX-114). The pellets from the  TX-114 extraction were then  resuspended in PBS-EDTA,  1% Triton X-100 by sonication, and warmed up to 37°C  for 15 min. The supernatants  (lanes E–H; TX-100) and pellets (lanes A–D; Pellet) of this  second extraction were also  run in SDS-PAGE. In all cases the total amount of protein was measured to ensure that all lanes for a given extraction procedure were  seeded with the same amounts of cellular material. All the lanes were blotted onto nitrocellulose sheets and probed with streptavidin–peroxidase and a chemiluminescence reaction. The small arrows between K and L point at apical bands now appearing also in the basolateral labeled set of proteins. The arrowheads indicate the position of molecular weight standards: (lanes A–D) 193, 112, 86, 70, 57, and 36  kD; (lanes E–L) 205, 116, 66, 45, and 29 kD. All the blots are from the same experiment, although, for technical reasons, they were run  in separate gels with two different sets of molecular weight standards. Biotinylation control: CACO-2 C2BBe monolayers were grown  on filters, incubated in A19, and biotinylated as described above. The cells were extensively washed, fixed in PFA, and processed with  fluorescein-coupled streptavidin from both sides of the filter. Optical confocal sections were taken at the transnuclear plane (a) or at the  apical membrane plane (b). Bars, 10 μm.
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Figure 9: Effect of antisense A19 oligonucleotide on the polarity of plasma membrane proteins in CACO-2 cells C2BBe1. The cells were continuously grown in random (lanes A, B, E, F, I, and J; control) or antisense A19 (lanes C, D, G, H, K, and L) oligonucleotides. For these experiments, the cells were plated on 24-mm TranswellTM filters and cultured at confluency for 9 d. The monolayers were biotinylated from either the apical side (lanes A, C, E, G, I, K; Ap) or from the basolateral side (lanes B, D, F, H, J, L; Bl). Then, the filters were extracted in ice-cold PBS-EDTA supplemented with 2% Triton X-114. The supernatant of this extraction was warmed to 30°C for 3 min, and the detergent phase was acetone precipitated and run in SDS-PAGE (lanes I–L; TX-114). The pellets from the TX-114 extraction were then resuspended in PBS-EDTA, 1% Triton X-100 by sonication, and warmed up to 37°C for 15 min. The supernatants (lanes E–H; TX-100) and pellets (lanes A–D; Pellet) of this second extraction were also run in SDS-PAGE. In all cases the total amount of protein was measured to ensure that all lanes for a given extraction procedure were seeded with the same amounts of cellular material. All the lanes were blotted onto nitrocellulose sheets and probed with streptavidin–peroxidase and a chemiluminescence reaction. The small arrows between K and L point at apical bands now appearing also in the basolateral labeled set of proteins. The arrowheads indicate the position of molecular weight standards: (lanes A–D) 193, 112, 86, 70, 57, and 36 kD; (lanes E–L) 205, 116, 66, 45, and 29 kD. All the blots are from the same experiment, although, for technical reasons, they were run in separate gels with two different sets of molecular weight standards. Biotinylation control: CACO-2 C2BBe monolayers were grown on filters, incubated in A19, and biotinylated as described above. The cells were extensively washed, fixed in PFA, and processed with fluorescein-coupled streptavidin from both sides of the filter. Optical confocal sections were taken at the transnuclear plane (a) or at the apical membrane plane (b). Bars, 10 μm.
Mentions: The cells were extracted at 0°C in Triton X-114, and the proteins were acetone precipitated from the 30°C detergent phase of the supernatant (Fig. 9, lanes I–L). These bands generally correspond to integral membrane proteins (Bordier, 1981). The pellets from the previous extraction were further extracted in Triton X-100 at 37°C. The proteins insoluble at 0–4°C but solubilized at a higher temperature typically correspond to (although perhaps are not exclusively) GPI-anchored membrane proteins (Brown and Rose, 1992; Fig. 9, lanes E–H). The results with this type of extraction were very similar to those obtained extracting the monolayers with 60 mM n-octyl-β-d-glycoside at 4°C after a Triton X-100 extraction at 0°C, another procedure to selectively extract GPI-anchored proteins (Hooper and Turner, 1988; Brown and Rose, 1992; and results not shown). Finally, the remaining membrane proteins in the pellet after both extractions are more likely to be truly associated with the submembrane cytoskeleton (Salas et al., 1988; Fig. 9, lanes A–D). Among the latter, the polarization was variable in control cells: at least five bands were distributed to both apical and basolateral domains, seven bands appeared mostly on the apical side, and 15–17 bands on the basolateral (Fig. 9, lanes A and B). A19 treated cells showed no detectable variations in the basolateral cytoskeletally associated membrane proteins (Fig. 9 D). The apical domain, on the other hand, displayed only three bands with levels of expression similar to the control (220, 194, and 72 kD). Two bands showed decreased levels (118 and 60 kD), while the rest were almost undetectable (Fig. 9 C). Coincidentally, the bands that were no longer expressed on the apical domain of A19 treated cells are in the same molecular weight range (40–86 kD) as those apical membrane proteins attached to CK19 multiprotein complexes in MDCK cells (Rodriguez et al., 1994). The bands extracted by Triton X-100 at 37°C after a previous extraction at 0°C showed a completely different pattern. As expected for GPI-anchored proteins (Lisanti et al., 1988), a vast majority of them localized to the apical domain in control cells (Fig. 9 E). In antisense treated cells, conversely, a number of bands appeared in the basolateral membrane (Fig. 9 H). Finally, the proteins extracted and purified in the detergent phase of Triton X-114 (integral membrane proteins; Bordier, 1981) showed a more complex pattern, with a number of bands polarized in either the apical or basolateral domains and at least four bands with the same molecular weights, presumably the same nonpolarized proteins, in both domains in control cells (Fig. 9, lanes I and J). Cells continuously grown in the presence of A19, however, showed five of the apical proteins redistributed to basolateral set (Fig. 9, marked by thin arrows between lanes K and L). Some proteins that remained nearly unchanged by the treatment with A19 have been marked with *. These examples, present in all lanes, rule out the possibility of variations in the efficiency of biotinylation.

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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