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AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture.

Benaud C, Gentil BJ, Assard N, Court M, Garin J, Delphin C, Baudier J - J. Cell Biol. (2003)

Bottom Line: Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane.In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height.We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

View Article: PubMed Central - PubMed

Affiliation: INSERM EMI-0104, DRDC-TS, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.

ABSTRACT
Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell-cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca(2+)-dependent cell-cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

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AHNAK colocalizes with the annexin 2/S100A10 complex at the plasma membrane in MDCK cells. (A) Confocal microscopy analysis (x-z axis) of the distribution in confluent MDCK cells of AHNAK (a), annexin 2 (b), and merge image (c). (B) Disruption of calcium-dependent cell–cell contacts causes the dissociation of AHNAK and annexin 2/S100A10 from the plasma membrane. Confluent MDCK cells were incubated in medium containing 5 mM EGTA supplemented with 1 mM MgCl2 for 30 min at 37°C (a and b), and were then shifted to calcium-containing medium for 30 min (c and d) or 3 h (e and f). Cultures were double immunolabeled for AHNAK (a, c, and e) and S100A10 (b, d, and f). Single-plane confocal microscopy (x-y axis) and x-z section (bottom) images are shown. (C) Confluent MDCK cells incubated with EGTA-MgCl2 medium for 30 min (lanes 1–2), then shifted back to Ca2+-containing medium for 3 h (lanes 3 and 4), were cross-linked with dithiobis succinimidyl propionate. Whole-cell lysates were incubated with protein A–Sepharose beads (lanes 1 and 3) or anti-AHNAK-CQL antibody cross-linked onto Sepharose beads (lanes 2 and 4). Immunoprecipitated proteins were reduced and analyzed by Western blot with anti-AHNAK-CQL, anti-actin, mouse monoclonal anti-annexin 2, and anti-S100A10 antibodies. (D) Confocal immunofluorescence of actin stained with phalloidin (a) and AHNAK (b) in MDCK cells treated with 1 μg/ml cytochalasin D for 1 h (c) is the merged image.
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fig3: AHNAK colocalizes with the annexin 2/S100A10 complex at the plasma membrane in MDCK cells. (A) Confocal microscopy analysis (x-z axis) of the distribution in confluent MDCK cells of AHNAK (a), annexin 2 (b), and merge image (c). (B) Disruption of calcium-dependent cell–cell contacts causes the dissociation of AHNAK and annexin 2/S100A10 from the plasma membrane. Confluent MDCK cells were incubated in medium containing 5 mM EGTA supplemented with 1 mM MgCl2 for 30 min at 37°C (a and b), and were then shifted to calcium-containing medium for 30 min (c and d) or 3 h (e and f). Cultures were double immunolabeled for AHNAK (a, c, and e) and S100A10 (b, d, and f). Single-plane confocal microscopy (x-y axis) and x-z section (bottom) images are shown. (C) Confluent MDCK cells incubated with EGTA-MgCl2 medium for 30 min (lanes 1–2), then shifted back to Ca2+-containing medium for 3 h (lanes 3 and 4), were cross-linked with dithiobis succinimidyl propionate. Whole-cell lysates were incubated with protein A–Sepharose beads (lanes 1 and 3) or anti-AHNAK-CQL antibody cross-linked onto Sepharose beads (lanes 2 and 4). Immunoprecipitated proteins were reduced and analyzed by Western blot with anti-AHNAK-CQL, anti-actin, mouse monoclonal anti-annexin 2, and anti-S100A10 antibodies. (D) Confocal immunofluorescence of actin stained with phalloidin (a) and AHNAK (b) in MDCK cells treated with 1 μg/ml cytochalasin D for 1 h (c) is the merged image.

Mentions: Confocal scanning immunofluorescence analyses of confluent epithelial MDCK cells reveal that AHNAK and annexin 2 colocalize all over the plasma membrane, including the sites of cell–cell contacts (Fig. 3 A). An identical pattern of distribution was observed for S100A10 (unpublished data). We had observed that AHNAK was recruited to the plasma membrane as cell confluence increased and as cells polarized (Fig. 1 A, e). The polarized phenotype of these cells was confirmed by the reorganization of the actin cytoskeleton (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200307098/DC1) and the apicolateral polarization of the tight junction marker ZO-1 (unpublished data). In polarized epithelial cells, cell–cell contacts initiate plasma membrane remodelling and the development of cell polarity (Drubin and Nelson, 1996). Therefore, we tested the role of the formation of cell–cell contacts in recruiting AHNAK to the plasma membrane. Lowering the Ca2+ concentration in the culture medium of an MDCK monolayer interferes with the stability of intercellular contacts (Grindstaff et al., 1998). Treatment of confluent MDCK cells with low Ca2+ medium resulted in the redistribution of AHNAK and of the annexin 2/S100A10 complex from cell contacts to the cytoplasm, and correlated with a more flattened morphology of the cells (Fig. 3 B, a and b). This membrane dissociation of AHNAK and of the annexin 2/S100A10 complex was reversible (Fig. 3 B, c–f). When cell–cell contacts were allowed to reform by readdition of calcium into the culture medium, AHNAK and the annexin 2/S100A10 complex were recruited to the sites of cell–cell contacts with a similar kinetic of relocation and distribution pattern. Within 30 min of calcium addition, AHNAK and S100A10 started to relocate to the newly forming cell–cell contacts (Fig. 3 B, c and d), and to strongly accumulate there after 3 h, as cell acquired a more cuboidal epithelial morphology (Fig. 3 B, e and f). Coimmunoprecipitation experiments on cross-linked MDCK cells during the calcium switch experiment revealed that AHNAK forms a multimeric complex containing actin and annexin 2/S100A10, and that this association is strictly dependent on the localization of AHNAK at the plasma membrane (Fig. 3 C, lane 4). Upon membrane dissociation of AHNAK, only S100A10, and to a lesser extent annexin 2, are recovered within the AHNAK immunoprecipitates (lane 2).


AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture.

Benaud C, Gentil BJ, Assard N, Court M, Garin J, Delphin C, Baudier J - J. Cell Biol. (2003)

AHNAK colocalizes with the annexin 2/S100A10 complex at the plasma membrane in MDCK cells. (A) Confocal microscopy analysis (x-z axis) of the distribution in confluent MDCK cells of AHNAK (a), annexin 2 (b), and merge image (c). (B) Disruption of calcium-dependent cell–cell contacts causes the dissociation of AHNAK and annexin 2/S100A10 from the plasma membrane. Confluent MDCK cells were incubated in medium containing 5 mM EGTA supplemented with 1 mM MgCl2 for 30 min at 37°C (a and b), and were then shifted to calcium-containing medium for 30 min (c and d) or 3 h (e and f). Cultures were double immunolabeled for AHNAK (a, c, and e) and S100A10 (b, d, and f). Single-plane confocal microscopy (x-y axis) and x-z section (bottom) images are shown. (C) Confluent MDCK cells incubated with EGTA-MgCl2 medium for 30 min (lanes 1–2), then shifted back to Ca2+-containing medium for 3 h (lanes 3 and 4), were cross-linked with dithiobis succinimidyl propionate. Whole-cell lysates were incubated with protein A–Sepharose beads (lanes 1 and 3) or anti-AHNAK-CQL antibody cross-linked onto Sepharose beads (lanes 2 and 4). Immunoprecipitated proteins were reduced and analyzed by Western blot with anti-AHNAK-CQL, anti-actin, mouse monoclonal anti-annexin 2, and anti-S100A10 antibodies. (D) Confocal immunofluorescence of actin stained with phalloidin (a) and AHNAK (b) in MDCK cells treated with 1 μg/ml cytochalasin D for 1 h (c) is the merged image.
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fig3: AHNAK colocalizes with the annexin 2/S100A10 complex at the plasma membrane in MDCK cells. (A) Confocal microscopy analysis (x-z axis) of the distribution in confluent MDCK cells of AHNAK (a), annexin 2 (b), and merge image (c). (B) Disruption of calcium-dependent cell–cell contacts causes the dissociation of AHNAK and annexin 2/S100A10 from the plasma membrane. Confluent MDCK cells were incubated in medium containing 5 mM EGTA supplemented with 1 mM MgCl2 for 30 min at 37°C (a and b), and were then shifted to calcium-containing medium for 30 min (c and d) or 3 h (e and f). Cultures were double immunolabeled for AHNAK (a, c, and e) and S100A10 (b, d, and f). Single-plane confocal microscopy (x-y axis) and x-z section (bottom) images are shown. (C) Confluent MDCK cells incubated with EGTA-MgCl2 medium for 30 min (lanes 1–2), then shifted back to Ca2+-containing medium for 3 h (lanes 3 and 4), were cross-linked with dithiobis succinimidyl propionate. Whole-cell lysates were incubated with protein A–Sepharose beads (lanes 1 and 3) or anti-AHNAK-CQL antibody cross-linked onto Sepharose beads (lanes 2 and 4). Immunoprecipitated proteins were reduced and analyzed by Western blot with anti-AHNAK-CQL, anti-actin, mouse monoclonal anti-annexin 2, and anti-S100A10 antibodies. (D) Confocal immunofluorescence of actin stained with phalloidin (a) and AHNAK (b) in MDCK cells treated with 1 μg/ml cytochalasin D for 1 h (c) is the merged image.
Mentions: Confocal scanning immunofluorescence analyses of confluent epithelial MDCK cells reveal that AHNAK and annexin 2 colocalize all over the plasma membrane, including the sites of cell–cell contacts (Fig. 3 A). An identical pattern of distribution was observed for S100A10 (unpublished data). We had observed that AHNAK was recruited to the plasma membrane as cell confluence increased and as cells polarized (Fig. 1 A, e). The polarized phenotype of these cells was confirmed by the reorganization of the actin cytoskeleton (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200307098/DC1) and the apicolateral polarization of the tight junction marker ZO-1 (unpublished data). In polarized epithelial cells, cell–cell contacts initiate plasma membrane remodelling and the development of cell polarity (Drubin and Nelson, 1996). Therefore, we tested the role of the formation of cell–cell contacts in recruiting AHNAK to the plasma membrane. Lowering the Ca2+ concentration in the culture medium of an MDCK monolayer interferes with the stability of intercellular contacts (Grindstaff et al., 1998). Treatment of confluent MDCK cells with low Ca2+ medium resulted in the redistribution of AHNAK and of the annexin 2/S100A10 complex from cell contacts to the cytoplasm, and correlated with a more flattened morphology of the cells (Fig. 3 B, a and b). This membrane dissociation of AHNAK and of the annexin 2/S100A10 complex was reversible (Fig. 3 B, c–f). When cell–cell contacts were allowed to reform by readdition of calcium into the culture medium, AHNAK and the annexin 2/S100A10 complex were recruited to the sites of cell–cell contacts with a similar kinetic of relocation and distribution pattern. Within 30 min of calcium addition, AHNAK and S100A10 started to relocate to the newly forming cell–cell contacts (Fig. 3 B, c and d), and to strongly accumulate there after 3 h, as cell acquired a more cuboidal epithelial morphology (Fig. 3 B, e and f). Coimmunoprecipitation experiments on cross-linked MDCK cells during the calcium switch experiment revealed that AHNAK forms a multimeric complex containing actin and annexin 2/S100A10, and that this association is strictly dependent on the localization of AHNAK at the plasma membrane (Fig. 3 C, lane 4). Upon membrane dissociation of AHNAK, only S100A10, and to a lesser extent annexin 2, are recovered within the AHNAK immunoprecipitates (lane 2).

Bottom Line: Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane.In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height.We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

View Article: PubMed Central - PubMed

Affiliation: INSERM EMI-0104, DRDC-TS, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.

ABSTRACT
Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell-cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca(2+)-dependent cell-cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

Show MeSH
Related in: MedlinePlus