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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  F-actin in CACO-2 cells. The  cells were continuously grown  in random (a, c, and e; control) or antisense A19 (b, d,  and f) oligonucleotides. For  this experiment, the cells were  plated on glass coverslips and  fixed in PFA after 9 d of confluency. The monolayers were  detergent permeabilized and  processed with FITC–phalloidin. The monolayers were  observed under a laser confocal microscope. Confocal  optical sections immediately  underneath the apical membrane (a and b) or immediately above the basal membrane (c and d) were chosen  from the stack of sections in  the z axis. The sections in a  and c correspond to the same  field at different focal planes,  and the same applies to the  sections in b and d. In each  field, a three-dimensional reconstruction section perpendicular to the plane of the  monolayer is shown in e and  f. Black arrowheads point at  F-actin negative apical regions in A19 treated cells.  Bars, 10 μm.
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Figure 6: Effect of antisense A19 oligonucleotide on the apical distribution of apical F-actin in CACO-2 cells. The cells were continuously grown in random (a, c, and e; control) or antisense A19 (b, d, and f) oligonucleotides. For this experiment, the cells were plated on glass coverslips and fixed in PFA after 9 d of confluency. The monolayers were detergent permeabilized and processed with FITC–phalloidin. The monolayers were observed under a laser confocal microscope. Confocal optical sections immediately underneath the apical membrane (a and b) or immediately above the basal membrane (c and d) were chosen from the stack of sections in the z axis. The sections in a and c correspond to the same field at different focal planes, and the same applies to the sections in b and d. In each field, a three-dimensional reconstruction section perpendicular to the plane of the monolayer is shown in e and f. Black arrowheads point at F-actin negative apical regions in A19 treated cells. Bars, 10 μm.

Mentions: Because the microvillus core contains F-actin, these results prompted us to study the distribution of F-actin in oligonucleotide-treated cells. Confocal optical sections of FITC–phalloidin stained cells at the apical level, under control oligonucleotides, displayed a typical punctate pattern in all cells (Fig. 6 a). In this case, occasional “black” or darker apical domains always corresponded to taller or smaller cells that became fully positive by adjusting the focal plane. In A19 treated monolayers on the other hand, 50– 70% of the cells displayed negative or low signal on the apical domains, contrasting with the still positive fluorescent rings at the cell–cell contacts. As an internal positive control, one cell that escaped the effect of A19 is shown on the upper right corner of Fig. 6 b. These positive cells were used as landmarks to determine the appropriate location of the otherwise negative apical surfaces in the z axis. Neither the submembrane F-actin under the lateral domains (not shown in x–y sections) nor the basal network of stress fibers (Fig. 6, c and d) showed any noticeable changes under the A19 treatment. Three-dimensional reconstructions in the x–z plane (perpendicular to the plane of the monolayer) showed a summary of the effect of CK19 antisense oligonucleotide: only apical F-actin was affected (compare Fig. 6 f, black arrowheads with control; e, white arrowheads point at the basal side). It must be noted that the total cellular levels of actin did not change in A19 treated cells (Fig. 4, lanes E and F), suggesting that the differences were in the polymerization and organization, not in the expression of actin. The correlation between the decrease in the number of microvilli and the effect of A19 was further analyzed by colocalization of villin and CK19 in A19 treated monolayers. In this experiment we analyzed the appearence of the typical apical image of fluorescence for villin in cells with or without CK19. For counting purposes, cells with reduced amounts of CK19 but still showing a CK19 IF network were ranked as positive for CK19. Only one of every seven cells negative to CK19 (CK19−) showed villin signal (Table II). On the other hand, 41% of the cells positive to CK19 ( CK19+) were also positive for villin (Table II). This experiment, in general, showed a good correlation between the loss of CK19 IF and the absence of microvilli. We cannot explain the 33% of the cells that do express CK19 and still lack microvilli, although it may be speculated that these cells may be delayed in their differentiation process.


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  F-actin in CACO-2 cells. The  cells were continuously grown  in random (a, c, and e; control) or antisense A19 (b, d,  and f) oligonucleotides. For  this experiment, the cells were  plated on glass coverslips and  fixed in PFA after 9 d of confluency. The monolayers were  detergent permeabilized and  processed with FITC–phalloidin. The monolayers were  observed under a laser confocal microscope. Confocal  optical sections immediately  underneath the apical membrane (a and b) or immediately above the basal membrane (c and d) were chosen  from the stack of sections in  the z axis. The sections in a  and c correspond to the same  field at different focal planes,  and the same applies to the  sections in b and d. In each  field, a three-dimensional reconstruction section perpendicular to the plane of the  monolayer is shown in e and  f. Black arrowheads point at  F-actin negative apical regions in A19 treated cells.  Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139782&req=5

Figure 6: Effect of antisense A19 oligonucleotide on the apical distribution of apical F-actin in CACO-2 cells. The cells were continuously grown in random (a, c, and e; control) or antisense A19 (b, d, and f) oligonucleotides. For this experiment, the cells were plated on glass coverslips and fixed in PFA after 9 d of confluency. The monolayers were detergent permeabilized and processed with FITC–phalloidin. The monolayers were observed under a laser confocal microscope. Confocal optical sections immediately underneath the apical membrane (a and b) or immediately above the basal membrane (c and d) were chosen from the stack of sections in the z axis. The sections in a and c correspond to the same field at different focal planes, and the same applies to the sections in b and d. In each field, a three-dimensional reconstruction section perpendicular to the plane of the monolayer is shown in e and f. Black arrowheads point at F-actin negative apical regions in A19 treated cells. Bars, 10 μm.
Mentions: Because the microvillus core contains F-actin, these results prompted us to study the distribution of F-actin in oligonucleotide-treated cells. Confocal optical sections of FITC–phalloidin stained cells at the apical level, under control oligonucleotides, displayed a typical punctate pattern in all cells (Fig. 6 a). In this case, occasional “black” or darker apical domains always corresponded to taller or smaller cells that became fully positive by adjusting the focal plane. In A19 treated monolayers on the other hand, 50– 70% of the cells displayed negative or low signal on the apical domains, contrasting with the still positive fluorescent rings at the cell–cell contacts. As an internal positive control, one cell that escaped the effect of A19 is shown on the upper right corner of Fig. 6 b. These positive cells were used as landmarks to determine the appropriate location of the otherwise negative apical surfaces in the z axis. Neither the submembrane F-actin under the lateral domains (not shown in x–y sections) nor the basal network of stress fibers (Fig. 6, c and d) showed any noticeable changes under the A19 treatment. Three-dimensional reconstructions in the x–z plane (perpendicular to the plane of the monolayer) showed a summary of the effect of CK19 antisense oligonucleotide: only apical F-actin was affected (compare Fig. 6 f, black arrowheads with control; e, white arrowheads point at the basal side). It must be noted that the total cellular levels of actin did not change in A19 treated cells (Fig. 4, lanes E and F), suggesting that the differences were in the polymerization and organization, not in the expression of actin. The correlation between the decrease in the number of microvilli and the effect of A19 was further analyzed by colocalization of villin and CK19 in A19 treated monolayers. In this experiment we analyzed the appearence of the typical apical image of fluorescence for villin in cells with or without CK19. For counting purposes, cells with reduced amounts of CK19 but still showing a CK19 IF network were ranked as positive for CK19. Only one of every seven cells negative to CK19 (CK19−) showed villin signal (Table II). On the other hand, 41% of the cells positive to CK19 ( CK19+) were also positive for villin (Table II). This experiment, in general, showed a good correlation between the loss of CK19 IF and the absence of microvilli. We cannot explain the 33% of the cells that do express CK19 and still lack microvilli, although it may be speculated that these cells may be delayed in their differentiation process.

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