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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 regulates actin-based cell membrane cytoarchitecture. (A) Down-regulation of AHNAK by siRNA in MDCK cells. Total cell lysates of cells treated with AHNAK RNA interference or control RNA interference were analyzed by Western blot for AHNAK, annexin 2, and actin levels. (B) Effect of AHNAK siRNA on actin organization. MDCK cells transfected with AHNAK RNA interference were stained for AHNAK (a–d) and F-actin (e–h) with phalloidin. Confocal x-y sections at the apical (a and e), upper lateral (b and f), lower lateral (c and g), and basal (d and h) levels of the cells show that cells devoid of AHNAK are unable to form an apicolateral cortical actin network, leaving a black void in the higher apicolateral planes. Rounded AHNAK-negative cells can be seen within the monolayer (arrows). (C) Confocal microscopy x-z merged sections of AHNAK (green) and actin (red) staining reveals that cells invalidated for AHNAK retains a flattened morphology. (D) The effect of AHNAK siRNA on basal actin stress fibers. Basal confocal microscopy section shows that cells invalidated for AHNAK (c and d) displayed disorganized actin stress fibers on their basal side, compared with control cells (a and b).
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fig7: AHNAK regulates actin-based cell membrane cytoarchitecture. (A) Down-regulation of AHNAK by siRNA in MDCK cells. Total cell lysates of cells treated with AHNAK RNA interference or control RNA interference were analyzed by Western blot for AHNAK, annexin 2, and actin levels. (B) Effect of AHNAK siRNA on actin organization. MDCK cells transfected with AHNAK RNA interference were stained for AHNAK (a–d) and F-actin (e–h) with phalloidin. Confocal x-y sections at the apical (a and e), upper lateral (b and f), lower lateral (c and g), and basal (d and h) levels of the cells show that cells devoid of AHNAK are unable to form an apicolateral cortical actin network, leaving a black void in the higher apicolateral planes. Rounded AHNAK-negative cells can be seen within the monolayer (arrows). (C) Confocal microscopy x-z merged sections of AHNAK (green) and actin (red) staining reveals that cells invalidated for AHNAK retains a flattened morphology. (D) The effect of AHNAK siRNA on basal actin stress fibers. Basal confocal microscopy section shows that cells invalidated for AHNAK (c and d) displayed disorganized actin stress fibers on their basal side, compared with control cells (a and b).

Mentions: To confirm the function of AHNAK in the regulation of cortical actin organization, we have down-regulated AHNAK levels in MDCK cells, which rearrange their cytoskeleton in a highly structured manner as they polarize concomitantly with AHNAK recruitment to the plasma membrane (Fig. 1). An siRNA designed against the highly conserved repeated central domains of AHNAK efficiently and specifically down-regulates AHNAK levels in MDCK cells (Fig. 7 A; Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200307098/DC1). We examined the effect of AHNAK siRNA on F-actin cytoskeleton rearrangement by confocal microscopy (Fig. 7, B–D). The extent of AHNAK down-regulation varied among transfected cells. Cells depleted for AHNAK were characterized by a marked disorganization of their actin cytoskeleton, visualized with fluorescent phalloidin. Unlike AHNAK-expressing cells, which rearranged their actin cytoskeleton into a lateral cortical belt and an apical actin network, cells devoid of AHNAK were unable to form an apicolateral cortical actin network, and F-actin could only be detected in the basal confocal plane (Fig. 7 B). AHNAK-negative cells retained a flattened morphology (Fig. 7 C). This decrease in cell height results in the void observed in the higher lateral and apical focal planes (Fig. 7 B, e and f). Note also that many AHNAK-negative cells at high density were dislodged from the cell monolayer and acquired rounded morphology (Fig. 7 B, arrows). This probably reflects an incapacity of invalidated cells to sustain density pressure because we did not observe a loss of adhesion of AHNAK-negative cells at low density (unpublished data). Although down-regulation of AHNAK has a drastic effect on actin cytoskeleton organization at the apicolateral plasma membrane that acts to support cell height, it has only a subtle effect on the basal actin organization. Cells invalidated for AHNAK only displayed more punctuate actin stress fibers on their basal side, compared with control cells (Fig. 7 D). Note also that in AHNAK-depleted cells, the annexin 2/S100A10 complex remains localized at the plasma membrane (unpublished data). This observation indicates that annexin 2/S100A10 is not sufficient to regulate actin cytoskeleton organization that acts to support cell height, and argues for a direct cooperation with AHNAK. Together, our data suggest that AHNAK regulates cortical actin cytoskeleton organization.


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 regulates actin-based cell membrane cytoarchitecture. (A) Down-regulation of AHNAK by siRNA in MDCK cells. Total cell lysates of cells treated with AHNAK RNA interference or control RNA interference were analyzed by Western blot for AHNAK, annexin 2, and actin levels. (B) Effect of AHNAK siRNA on actin organization. MDCK cells transfected with AHNAK RNA interference were stained for AHNAK (a–d) and F-actin (e–h) with phalloidin. Confocal x-y sections at the apical (a and e), upper lateral (b and f), lower lateral (c and g), and basal (d and h) levels of the cells show that cells devoid of AHNAK are unable to form an apicolateral cortical actin network, leaving a black void in the higher apicolateral planes. Rounded AHNAK-negative cells can be seen within the monolayer (arrows). (C) Confocal microscopy x-z merged sections of AHNAK (green) and actin (red) staining reveals that cells invalidated for AHNAK retains a flattened morphology. (D) The effect of AHNAK siRNA on basal actin stress fibers. Basal confocal microscopy section shows that cells invalidated for AHNAK (c and d) displayed disorganized actin stress fibers on their basal side, compared with control cells (a and b).
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Related In: Results  -  Collection

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fig7: AHNAK regulates actin-based cell membrane cytoarchitecture. (A) Down-regulation of AHNAK by siRNA in MDCK cells. Total cell lysates of cells treated with AHNAK RNA interference or control RNA interference were analyzed by Western blot for AHNAK, annexin 2, and actin levels. (B) Effect of AHNAK siRNA on actin organization. MDCK cells transfected with AHNAK RNA interference were stained for AHNAK (a–d) and F-actin (e–h) with phalloidin. Confocal x-y sections at the apical (a and e), upper lateral (b and f), lower lateral (c and g), and basal (d and h) levels of the cells show that cells devoid of AHNAK are unable to form an apicolateral cortical actin network, leaving a black void in the higher apicolateral planes. Rounded AHNAK-negative cells can be seen within the monolayer (arrows). (C) Confocal microscopy x-z merged sections of AHNAK (green) and actin (red) staining reveals that cells invalidated for AHNAK retains a flattened morphology. (D) The effect of AHNAK siRNA on basal actin stress fibers. Basal confocal microscopy section shows that cells invalidated for AHNAK (c and d) displayed disorganized actin stress fibers on their basal side, compared with control cells (a and b).
Mentions: To confirm the function of AHNAK in the regulation of cortical actin organization, we have down-regulated AHNAK levels in MDCK cells, which rearrange their cytoskeleton in a highly structured manner as they polarize concomitantly with AHNAK recruitment to the plasma membrane (Fig. 1). An siRNA designed against the highly conserved repeated central domains of AHNAK efficiently and specifically down-regulates AHNAK levels in MDCK cells (Fig. 7 A; Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200307098/DC1). We examined the effect of AHNAK siRNA on F-actin cytoskeleton rearrangement by confocal microscopy (Fig. 7, B–D). The extent of AHNAK down-regulation varied among transfected cells. Cells depleted for AHNAK were characterized by a marked disorganization of their actin cytoskeleton, visualized with fluorescent phalloidin. Unlike AHNAK-expressing cells, which rearranged their actin cytoskeleton into a lateral cortical belt and an apical actin network, cells devoid of AHNAK were unable to form an apicolateral cortical actin network, and F-actin could only be detected in the basal confocal plane (Fig. 7 B). AHNAK-negative cells retained a flattened morphology (Fig. 7 C). This decrease in cell height results in the void observed in the higher lateral and apical focal planes (Fig. 7 B, e and f). Note also that many AHNAK-negative cells at high density were dislodged from the cell monolayer and acquired rounded morphology (Fig. 7 B, arrows). This probably reflects an incapacity of invalidated cells to sustain density pressure because we did not observe a loss of adhesion of AHNAK-negative cells at low density (unpublished data). Although down-regulation of AHNAK has a drastic effect on actin cytoskeleton organization at the apicolateral plasma membrane that acts to support cell height, it has only a subtle effect on the basal actin organization. Cells invalidated for AHNAK only displayed more punctuate actin stress fibers on their basal side, compared with control cells (Fig. 7 D). Note also that in AHNAK-depleted cells, the annexin 2/S100A10 complex remains localized at the plasma membrane (unpublished data). This observation indicates that annexin 2/S100A10 is not sufficient to regulate actin cytoskeleton organization that acts to support cell height, and argues for a direct cooperation with AHNAK. Together, our data suggest that AHNAK regulates cortical actin cytoskeleton organization.

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