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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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The annexin 2/S100A10 complex mediates AHNAK targeting to the cell plasma membrane in MCF-7 cells. (A) Western blot analysis of MCF-7 cell extracts after transfection with control RNA interference (lane 1) or annexin 2 siRNA (lane 2). Total protein extracts were probed by Western blot with anti-annexin 2, -4 and -6 antibodies (left) or with annexin 2, AHNAK, S100A10, and actin antibodies (right). (B) Confocal microscopy analysis of annexin 2 (a and c) and AHNAK (b, d, and e) in MCF-7 cells transfected with control RNA interference (a and b) or annexin 2 siRNA (c–e). The cytoplasmic localization of AHNAK in annexin 2–depleted cells is seen in x-y (d) and x-z sections (c). (C) Down-regulation of annexin 2/S100A10 prevents calcium-dependent relocation of AHNAK to the plasma membrane. Confocal microscopy analysis of S100A10 (a, c, and e) and AHNAK (b, d, f) distribution in control MCF-7 cells (a–d), and in MCF-7 transfected with annexin 2 siRNA (e and f). MCF-7 cells were incubated in EGTA-MgCl2 medium for 30 min (a and b), then shifted to Ca2+-containing medium for 3 h (c–f). Both x-y and x-z sections are shown.
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fig5: The annexin 2/S100A10 complex mediates AHNAK targeting to the cell plasma membrane in MCF-7 cells. (A) Western blot analysis of MCF-7 cell extracts after transfection with control RNA interference (lane 1) or annexin 2 siRNA (lane 2). Total protein extracts were probed by Western blot with anti-annexin 2, -4 and -6 antibodies (left) or with annexin 2, AHNAK, S100A10, and actin antibodies (right). (B) Confocal microscopy analysis of annexin 2 (a and c) and AHNAK (b, d, and e) in MCF-7 cells transfected with control RNA interference (a and b) or annexin 2 siRNA (c–e). The cytoplasmic localization of AHNAK in annexin 2–depleted cells is seen in x-y (d) and x-z sections (c). (C) Down-regulation of annexin 2/S100A10 prevents calcium-dependent relocation of AHNAK to the plasma membrane. Confocal microscopy analysis of S100A10 (a, c, and e) and AHNAK (b, d, f) distribution in control MCF-7 cells (a–d), and in MCF-7 transfected with annexin 2 siRNA (e and f). MCF-7 cells were incubated in EGTA-MgCl2 medium for 30 min (a and b), then shifted to Ca2+-containing medium for 3 h (c–f). Both x-y and x-z sections are shown.

Mentions: To evaluate whether the annexin 2/S100A10 complex cooperates with AHNAK functions at the plasma membrane, we first analyzed the incidence of annexin 2 down-regulation on AHNAK subcellular localization. A small interfering RNA (siRNA) duplex was designed against the human annexin 2 cDNA sequence. This siRNA does not function in down-regulating canine annexin 2 in MDCK cells, but is highly efficient in human epithelial MCF-7 cells (Fig. 5 A). Transfection of MCF-7 cells with annexin 2 siRNA significantly abrogated the expression of annexin 2, but did not affect the levels of related annexins expressed in MCF-7, annexin 4, and annexin 6. The down-regulation of annexin 2 in MCF-7 cells induced a drastic concomitant down-regulation of S100A10. This was likely the result of both transcriptional and post-translational regulations of the S100A10 level by annexin 2, a previously described well-known phenomenon (Puisieux et al., 1996; Van de Graaf et al., 2003). A decrease in AHNAK levels was also observed in transfected cells. This effect of annexin 2 RNA interference on AHNAK levels was specific because the total level of actin was not affected, and the transfection with a nonspecific siRNA did not alter the levels of AHNAK (unpublished data). Consistent with the Western blot data, the membrane-associated AHNAK immunoreactivity dropped in annexin 2–depleted MCF-7 cells, and the remaining AHNAK signal appeared diffuse within the cytoplasm (Fig. 5 B). The interdependence that exists between the expression of annexin 2/S100A10 and the recruitment of AHNAK at the plasma membrane was confirmed in Ca2+ switch experiments (Fig. 5 C). As observed with MDCK cells (Fig. 3 B), disruption of calcium-dependent cell–cell contacts causes a reversible dissociation of AHNAK and of the annexin 2/S100A10 complex from the plasma membrane in MCF-7 cells (Fig. 5 C, a–d). However, in cells depleted with annexin 2/S100A10, AHNAK could not be re-recruited to the plasma membrane upon Ca2+-mediated cell–cell adhesion, and cells retained a flattened morphology (Fig. 5 C, e and f).


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)

The annexin 2/S100A10 complex mediates AHNAK targeting to the cell plasma membrane in MCF-7 cells. (A) Western blot analysis of MCF-7 cell extracts after transfection with control RNA interference (lane 1) or annexin 2 siRNA (lane 2). Total protein extracts were probed by Western blot with anti-annexin 2, -4 and -6 antibodies (left) or with annexin 2, AHNAK, S100A10, and actin antibodies (right). (B) Confocal microscopy analysis of annexin 2 (a and c) and AHNAK (b, d, and e) in MCF-7 cells transfected with control RNA interference (a and b) or annexin 2 siRNA (c–e). The cytoplasmic localization of AHNAK in annexin 2–depleted cells is seen in x-y (d) and x-z sections (c). (C) Down-regulation of annexin 2/S100A10 prevents calcium-dependent relocation of AHNAK to the plasma membrane. Confocal microscopy analysis of S100A10 (a, c, and e) and AHNAK (b, d, f) distribution in control MCF-7 cells (a–d), and in MCF-7 transfected with annexin 2 siRNA (e and f). MCF-7 cells were incubated in EGTA-MgCl2 medium for 30 min (a and b), then shifted to Ca2+-containing medium for 3 h (c–f). Both x-y and x-z sections are shown.
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fig5: The annexin 2/S100A10 complex mediates AHNAK targeting to the cell plasma membrane in MCF-7 cells. (A) Western blot analysis of MCF-7 cell extracts after transfection with control RNA interference (lane 1) or annexin 2 siRNA (lane 2). Total protein extracts were probed by Western blot with anti-annexin 2, -4 and -6 antibodies (left) or with annexin 2, AHNAK, S100A10, and actin antibodies (right). (B) Confocal microscopy analysis of annexin 2 (a and c) and AHNAK (b, d, and e) in MCF-7 cells transfected with control RNA interference (a and b) or annexin 2 siRNA (c–e). The cytoplasmic localization of AHNAK in annexin 2–depleted cells is seen in x-y (d) and x-z sections (c). (C) Down-regulation of annexin 2/S100A10 prevents calcium-dependent relocation of AHNAK to the plasma membrane. Confocal microscopy analysis of S100A10 (a, c, and e) and AHNAK (b, d, f) distribution in control MCF-7 cells (a–d), and in MCF-7 transfected with annexin 2 siRNA (e and f). MCF-7 cells were incubated in EGTA-MgCl2 medium for 30 min (a and b), then shifted to Ca2+-containing medium for 3 h (c–f). Both x-y and x-z sections are shown.
Mentions: To evaluate whether the annexin 2/S100A10 complex cooperates with AHNAK functions at the plasma membrane, we first analyzed the incidence of annexin 2 down-regulation on AHNAK subcellular localization. A small interfering RNA (siRNA) duplex was designed against the human annexin 2 cDNA sequence. This siRNA does not function in down-regulating canine annexin 2 in MDCK cells, but is highly efficient in human epithelial MCF-7 cells (Fig. 5 A). Transfection of MCF-7 cells with annexin 2 siRNA significantly abrogated the expression of annexin 2, but did not affect the levels of related annexins expressed in MCF-7, annexin 4, and annexin 6. The down-regulation of annexin 2 in MCF-7 cells induced a drastic concomitant down-regulation of S100A10. This was likely the result of both transcriptional and post-translational regulations of the S100A10 level by annexin 2, a previously described well-known phenomenon (Puisieux et al., 1996; Van de Graaf et al., 2003). A decrease in AHNAK levels was also observed in transfected cells. This effect of annexin 2 RNA interference on AHNAK levels was specific because the total level of actin was not affected, and the transfection with a nonspecific siRNA did not alter the levels of AHNAK (unpublished data). Consistent with the Western blot data, the membrane-associated AHNAK immunoreactivity dropped in annexin 2–depleted MCF-7 cells, and the remaining AHNAK signal appeared diffuse within the cytoplasm (Fig. 5 B). The interdependence that exists between the expression of annexin 2/S100A10 and the recruitment of AHNAK at the plasma membrane was confirmed in Ca2+ switch experiments (Fig. 5 C). As observed with MDCK cells (Fig. 3 B), disruption of calcium-dependent cell–cell contacts causes a reversible dissociation of AHNAK and of the annexin 2/S100A10 complex from the plasma membrane in MCF-7 cells (Fig. 5 C, a–d). However, in cells depleted with annexin 2/S100A10, AHNAK could not be re-recruited to the plasma membrane upon Ca2+-mediated cell–cell adhesion, and cells retained a flattened morphology (Fig. 5 C, e and f).

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