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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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Immunoblot of cytoskeletal components in cells continuously kept in random (A, C, E, G, and I), A19 (B, D, F, H, and  J), random/2 (K, M, O, and Q), or A19/2 (L, N, P, and R) oligonucleotides. CACO-2 (A–F and K–N) and MCF-10A (G–J and  O–R) cells were grown on 24-mm Transwell™ filters and extracted/scrapped from the filter in EB-TX- 100. The pellets were  further extracted in SDS sample buffer, and samples from equivalent numbers of cells were run in SDS-PAGE ( the protein in  each sample was measured in a small aliquot and the sample volume adjusted to seed 4 μg of protein in all lanes; usually these adjustments represented variations in sample volumes that were  <10% of the total volume) and immunoblotted onto nitrocellulose filters. The blots were processed for indirect chemiluminescence using an anti-CK19 mAb (RCK108; A, B, G, and H). The  same nitrocellulose sheets used for A, B, G, H, K, L, O, and P were  then stripped in SDS/2-mercaptoethanol and reprobed with antiPancytokeratin mAb (AE1/AE3; C, D, I–J, M, N, and O–R). The  same filter from CACO-2 extract was stripped and reprobed one  more time with anti-actin mAb (C4; E and F). Notice the decrease in CK19 bands in A19 treated cells (B and H) as compared  with the control with random oligonucleotide (A and G), and that  other cytokeratins and actin in the same samples were not  changed (C–F, I, and J). The average OD measures obtained from  unfiltered digitized images after subtracting background from the  average pixel value over the main band of each lane were as follows (scale 0–255): (A) 82; (B) 12; (C) 77; (D) 100; (E) 91; (F) 81;  (G) 30; (H) 6; (I) 28; (J) 21; (K) 150; (L) 27; (M) 171; (N) 172; (O)  49, (P) 5; (Q) 160; (R) 174. The apparent molecular weight of  standards is expressed in kD.
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Figure 4: Immunoblot of cytoskeletal components in cells continuously kept in random (A, C, E, G, and I), A19 (B, D, F, H, and J), random/2 (K, M, O, and Q), or A19/2 (L, N, P, and R) oligonucleotides. CACO-2 (A–F and K–N) and MCF-10A (G–J and O–R) cells were grown on 24-mm Transwell™ filters and extracted/scrapped from the filter in EB-TX- 100. The pellets were further extracted in SDS sample buffer, and samples from equivalent numbers of cells were run in SDS-PAGE ( the protein in each sample was measured in a small aliquot and the sample volume adjusted to seed 4 μg of protein in all lanes; usually these adjustments represented variations in sample volumes that were <10% of the total volume) and immunoblotted onto nitrocellulose filters. The blots were processed for indirect chemiluminescence using an anti-CK19 mAb (RCK108; A, B, G, and H). The same nitrocellulose sheets used for A, B, G, H, K, L, O, and P were then stripped in SDS/2-mercaptoethanol and reprobed with antiPancytokeratin mAb (AE1/AE3; C, D, I–J, M, N, and O–R). The same filter from CACO-2 extract was stripped and reprobed one more time with anti-actin mAb (C4; E and F). Notice the decrease in CK19 bands in A19 treated cells (B and H) as compared with the control with random oligonucleotide (A and G), and that other cytokeratins and actin in the same samples were not changed (C–F, I, and J). The average OD measures obtained from unfiltered digitized images after subtracting background from the average pixel value over the main band of each lane were as follows (scale 0–255): (A) 82; (B) 12; (C) 77; (D) 100; (E) 91; (F) 81; (G) 30; (H) 6; (I) 28; (J) 21; (K) 150; (L) 27; (M) 171; (N) 172; (O) 49, (P) 5; (Q) 160; (R) 174. The apparent molecular weight of standards is expressed in kD.

Mentions: The effect of the antisense oligonucleotides was also determined by immunoblot. SDS extracts of cytoskeletal pellets from CACO-2 cells incubated in A19 showed a clear decrease in CK19 (Fig. 4, lane B, 37% of the signal in the band in lane A) as compared with controls kept in random oligonucleotide (Fig. 4, lane A). A similar effect was observed with A19/2 (Fig. 4, lanes K and L). When the same nitrocellulose sheet was stripped and reprobed with a mAb against other cytokeratins or a mAb against all actin isoforms, no differences were observed (Fig. 4, lanes C–F, M, and N), indicating that the effects of A19 and A19/2 were specific for CK19. Likewise, MCF-10A cells continuously grown in A19 displayed decreased levels of CK19 (Fig. 4, lane H) as compared with the controls in random oligonucleotide (Fig. 4, lane G), and a comparable effect of A19/2 (Fig. 4, O and P). Reprobing the same nitrocellulose sheet with the anti-pan cytokeratin mAb showed, again, no effect on other cytoskeletal proteins (Fig. 4, lanes I, J, Q, and R). These results were quantitatively determined by analysis of the digitized images from chemiluminescence detection (Fig. 4, legend). It must be pointed out that three to four bands were detected by RCK108 mAb in MCF10A cells, with apparent molecular weights ranging from 44 to 53 kD, in some preparations. Interestingly, the same pattern of bands was displayed by K4.62 mAb in some preparations from MDCK cells. Our polyclonal Ab consistently displayed affinity for the 53-kD band. We have no explanation for these multiple bands, although we speculate that different states of phosphorylation may be responsible for this type of pattern. On the other hand, it has been shown that mammary epithelia display a complex pattern of cytokeratins, including at least one that is induced by extracellular matrix (Hall and Bissell, 1986). Therefore, it is also possible that RCK108 may be recognizing an epitope in other cytokeratins from MCF-10A that are not present in CACO-2 cells. If that was the case, these other cytokeratins may also share a common 5′ sequence in the reading frame of their respective mRNAs, since they were also downregulated by A19.


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)

Immunoblot of cytoskeletal components in cells continuously kept in random (A, C, E, G, and I), A19 (B, D, F, H, and  J), random/2 (K, M, O, and Q), or A19/2 (L, N, P, and R) oligonucleotides. CACO-2 (A–F and K–N) and MCF-10A (G–J and  O–R) cells were grown on 24-mm Transwell™ filters and extracted/scrapped from the filter in EB-TX- 100. The pellets were  further extracted in SDS sample buffer, and samples from equivalent numbers of cells were run in SDS-PAGE ( the protein in  each sample was measured in a small aliquot and the sample volume adjusted to seed 4 μg of protein in all lanes; usually these adjustments represented variations in sample volumes that were  <10% of the total volume) and immunoblotted onto nitrocellulose filters. The blots were processed for indirect chemiluminescence using an anti-CK19 mAb (RCK108; A, B, G, and H). The  same nitrocellulose sheets used for A, B, G, H, K, L, O, and P were  then stripped in SDS/2-mercaptoethanol and reprobed with antiPancytokeratin mAb (AE1/AE3; C, D, I–J, M, N, and O–R). The  same filter from CACO-2 extract was stripped and reprobed one  more time with anti-actin mAb (C4; E and F). Notice the decrease in CK19 bands in A19 treated cells (B and H) as compared  with the control with random oligonucleotide (A and G), and that  other cytokeratins and actin in the same samples were not  changed (C–F, I, and J). The average OD measures obtained from  unfiltered digitized images after subtracting background from the  average pixel value over the main band of each lane were as follows (scale 0–255): (A) 82; (B) 12; (C) 77; (D) 100; (E) 91; (F) 81;  (G) 30; (H) 6; (I) 28; (J) 21; (K) 150; (L) 27; (M) 171; (N) 172; (O)  49, (P) 5; (Q) 160; (R) 174. The apparent molecular weight of  standards is expressed in kD.
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Figure 4: Immunoblot of cytoskeletal components in cells continuously kept in random (A, C, E, G, and I), A19 (B, D, F, H, and J), random/2 (K, M, O, and Q), or A19/2 (L, N, P, and R) oligonucleotides. CACO-2 (A–F and K–N) and MCF-10A (G–J and O–R) cells were grown on 24-mm Transwell™ filters and extracted/scrapped from the filter in EB-TX- 100. The pellets were further extracted in SDS sample buffer, and samples from equivalent numbers of cells were run in SDS-PAGE ( the protein in each sample was measured in a small aliquot and the sample volume adjusted to seed 4 μg of protein in all lanes; usually these adjustments represented variations in sample volumes that were <10% of the total volume) and immunoblotted onto nitrocellulose filters. The blots were processed for indirect chemiluminescence using an anti-CK19 mAb (RCK108; A, B, G, and H). The same nitrocellulose sheets used for A, B, G, H, K, L, O, and P were then stripped in SDS/2-mercaptoethanol and reprobed with antiPancytokeratin mAb (AE1/AE3; C, D, I–J, M, N, and O–R). The same filter from CACO-2 extract was stripped and reprobed one more time with anti-actin mAb (C4; E and F). Notice the decrease in CK19 bands in A19 treated cells (B and H) as compared with the control with random oligonucleotide (A and G), and that other cytokeratins and actin in the same samples were not changed (C–F, I, and J). The average OD measures obtained from unfiltered digitized images after subtracting background from the average pixel value over the main band of each lane were as follows (scale 0–255): (A) 82; (B) 12; (C) 77; (D) 100; (E) 91; (F) 81; (G) 30; (H) 6; (I) 28; (J) 21; (K) 150; (L) 27; (M) 171; (N) 172; (O) 49, (P) 5; (Q) 160; (R) 174. The apparent molecular weight of standards is expressed in kD.
Mentions: The effect of the antisense oligonucleotides was also determined by immunoblot. SDS extracts of cytoskeletal pellets from CACO-2 cells incubated in A19 showed a clear decrease in CK19 (Fig. 4, lane B, 37% of the signal in the band in lane A) as compared with controls kept in random oligonucleotide (Fig. 4, lane A). A similar effect was observed with A19/2 (Fig. 4, lanes K and L). When the same nitrocellulose sheet was stripped and reprobed with a mAb against other cytokeratins or a mAb against all actin isoforms, no differences were observed (Fig. 4, lanes C–F, M, and N), indicating that the effects of A19 and A19/2 were specific for CK19. Likewise, MCF-10A cells continuously grown in A19 displayed decreased levels of CK19 (Fig. 4, lane H) as compared with the controls in random oligonucleotide (Fig. 4, lane G), and a comparable effect of A19/2 (Fig. 4, O and P). Reprobing the same nitrocellulose sheet with the anti-pan cytokeratin mAb showed, again, no effect on other cytoskeletal proteins (Fig. 4, lanes I, J, Q, and R). These results were quantitatively determined by analysis of the digitized images from chemiluminescence detection (Fig. 4, legend). It must be pointed out that three to four bands were detected by RCK108 mAb in MCF10A cells, with apparent molecular weights ranging from 44 to 53 kD, in some preparations. Interestingly, the same pattern of bands was displayed by K4.62 mAb in some preparations from MDCK cells. Our polyclonal Ab consistently displayed affinity for the 53-kD band. We have no explanation for these multiple bands, although we speculate that different states of phosphorylation may be responsible for this type of pattern. On the other hand, it has been shown that mammary epithelia display a complex pattern of cytokeratins, including at least one that is induced by extracellular matrix (Hall and Bissell, 1986). Therefore, it is also possible that RCK108 may be recognizing an epitope in other cytokeratins from MCF-10A that are not present in CACO-2 cells. If that was the case, these other cytokeratins may also share a common 5′ sequence in the reading frame of their respective mRNAs, since they were also downregulated by A19.

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