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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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Density-dependent localization of AHNAK to the cytoplasmic face of the MDCK cell plasma membrane. (A) Redistribution of AHNAK and actin during confluence mediated cell–cell contact and polarization. MDCK cells were cultured on glass coverslips for 2 (a and b), 24 (c and d) or 96 h (e and f), and stained with anti-AHNAK-KIS antibody (a, c, and e) and F-actin visualized with phalloidin (b, d, and f). (B) Confocal immunofluorescence with polyclonal anti-AHNAK-KIS antibody of streptolysin O–permeabilized confluent MDCK cells reveals a positive signal at the cytoplasmic surface of the plasma membrane.
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fig1: Density-dependent localization of AHNAK to the cytoplasmic face of the MDCK cell plasma membrane. (A) Redistribution of AHNAK and actin during confluence mediated cell–cell contact and polarization. MDCK cells were cultured on glass coverslips for 2 (a and b), 24 (c and d) or 96 h (e and f), and stained with anti-AHNAK-KIS antibody (a, c, and e) and F-actin visualized with phalloidin (b, d, and f). (B) Confocal immunofluorescence with polyclonal anti-AHNAK-KIS antibody of streptolysin O–permeabilized confluent MDCK cells reveals a positive signal at the cytoplasmic surface of the plasma membrane.

Mentions: In canine epithelial MDCK cells, AHNAK redistributes from the cytoplasm to a cortical/plasma membrane localization as cell density increases and as cells establish cell–cell contacts (Sussman et al., 2001; Fig. 1 A). This redistribution of AHNAK follows the reorganization of the actin cytoskeleton into cortical actin (Fig. 1 A). Three different AHNAK antibodies, the affinity-purified AHNAK-KIS pAb directed against the repeated central domain of AHNAK, the affinity-purified AHNAK-CQL pAb directed against the extreme COOH terminus (Gentil et al., 2003), and the anti-AHNAK (desmoyokin) antibody (DY) (Hashimoto et al., 1993) gave a similar immunostaining pattern. A recent work has shown that in secretory cell models, AHNAK can be localized within the lumen of specific vesicles called enlargosomes and is redistributed to the external surface of the plasma membrane in response to large increases in Ca2+ (Borgonovo et al., 2002). Therefore, we evaluated in confluent MDCK cells on which side of the plasma membrane AHNAK is distributed, and whether AHNAK is present in the lumen of cortical vesicles or in the cytoplasm. To resolve this issue, we compared the immunostaining obtained with our affinity-purified anti-AHNAK-KIS antibody in detergent-permeabilized cells, in streptolysin O–permeabilized cells, and in nonpermeabilized live confluent MDCK cells. In nonpermeabilized cells, no surface AHNAK immunolabeling was detected (unpublished data). When the plasma membrane of live cells was permeabilized with streptolysin O, a condition in which only cytoplasmic and not the vesicle luminal proteins are accessible to the antibodies, the plasma membrane was labeled (Fig. 1 B). An identical result was obtained with the anti-AHNAK (desmoyokin) antibody (DY) previously shown to recognize the membrane-bound protein in epithelial cells (Hashimoto et al., 1993, 1995; unpublished data). Even though we cannot exclude the possibility that a population of AHNAK is present in enlargosomes, these results clearly indicate that in our epithelial cell model, AHNAK is predominantly a cytosolic protein localized at the intracellular face of plasma membrane.


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)

Density-dependent localization of AHNAK to the cytoplasmic face of the MDCK cell plasma membrane. (A) Redistribution of AHNAK and actin during confluence mediated cell–cell contact and polarization. MDCK cells were cultured on glass coverslips for 2 (a and b), 24 (c and d) or 96 h (e and f), and stained with anti-AHNAK-KIS antibody (a, c, and e) and F-actin visualized with phalloidin (b, d, and f). (B) Confocal immunofluorescence with polyclonal anti-AHNAK-KIS antibody of streptolysin O–permeabilized confluent MDCK cells reveals a positive signal at the cytoplasmic surface of the plasma membrane.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171952&req=5

fig1: Density-dependent localization of AHNAK to the cytoplasmic face of the MDCK cell plasma membrane. (A) Redistribution of AHNAK and actin during confluence mediated cell–cell contact and polarization. MDCK cells were cultured on glass coverslips for 2 (a and b), 24 (c and d) or 96 h (e and f), and stained with anti-AHNAK-KIS antibody (a, c, and e) and F-actin visualized with phalloidin (b, d, and f). (B) Confocal immunofluorescence with polyclonal anti-AHNAK-KIS antibody of streptolysin O–permeabilized confluent MDCK cells reveals a positive signal at the cytoplasmic surface of the plasma membrane.
Mentions: In canine epithelial MDCK cells, AHNAK redistributes from the cytoplasm to a cortical/plasma membrane localization as cell density increases and as cells establish cell–cell contacts (Sussman et al., 2001; Fig. 1 A). This redistribution of AHNAK follows the reorganization of the actin cytoskeleton into cortical actin (Fig. 1 A). Three different AHNAK antibodies, the affinity-purified AHNAK-KIS pAb directed against the repeated central domain of AHNAK, the affinity-purified AHNAK-CQL pAb directed against the extreme COOH terminus (Gentil et al., 2003), and the anti-AHNAK (desmoyokin) antibody (DY) (Hashimoto et al., 1993) gave a similar immunostaining pattern. A recent work has shown that in secretory cell models, AHNAK can be localized within the lumen of specific vesicles called enlargosomes and is redistributed to the external surface of the plasma membrane in response to large increases in Ca2+ (Borgonovo et al., 2002). Therefore, we evaluated in confluent MDCK cells on which side of the plasma membrane AHNAK is distributed, and whether AHNAK is present in the lumen of cortical vesicles or in the cytoplasm. To resolve this issue, we compared the immunostaining obtained with our affinity-purified anti-AHNAK-KIS antibody in detergent-permeabilized cells, in streptolysin O–permeabilized cells, and in nonpermeabilized live confluent MDCK cells. In nonpermeabilized cells, no surface AHNAK immunolabeling was detected (unpublished data). When the plasma membrane of live cells was permeabilized with streptolysin O, a condition in which only cytoplasmic and not the vesicle luminal proteins are accessible to the antibodies, the plasma membrane was labeled (Fig. 1 B). An identical result was obtained with the anti-AHNAK (desmoyokin) antibody (DY) previously shown to recognize the membrane-bound protein in epithelial cells (Hashimoto et al., 1993, 1995; unpublished data). Even though we cannot exclude the possibility that a population of AHNAK is present in enlargosomes, these results clearly indicate that in our epithelial cell model, AHNAK is predominantly a cytosolic protein localized at the intracellular face of plasma membrane.

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