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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 and annexin 2/S100A10 associate with cholesterol-rich membranes. (A) AHNAK and annexin 2/S100A10 cofractionate to the lipid raft fractions. MDCK cells were lysed in buffer containing 1% Triton X-100 on ice, and flotation fractionation was performed in a 5–40% OptiPrep™ gradient. Fractions were analyzed by Western blotting using antibodies against AHNAK, annexin 2, and S100A10. Caveolin-1 was used as a marker of lipid raft–containing fraction. (B) Confocal microscopy analysis of FITC-cholera toxin β chain (a) and AHNAK (b) colocalization in MDCK cells. (c) Merged image. (C) Specific release of membrane-bound AHNAK by sequestration of membrane cholesterol. Confocal microscopy analysis of AHNAK (a, b, e, and f) and annexin 2 (HH7; c, d, g, and h) in MCF-7 (a–d) and MDCK (e–h) cells not treated (a, c, e, and g) or incubated with 3.8 mM of methyl-β-cyclodextrin (MβCD) for 15 min (b and d) or 1 h (f and h) at 37°C before fixation.
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fig4: AHNAK and annexin 2/S100A10 associate with cholesterol-rich membranes. (A) AHNAK and annexin 2/S100A10 cofractionate to the lipid raft fractions. MDCK cells were lysed in buffer containing 1% Triton X-100 on ice, and flotation fractionation was performed in a 5–40% OptiPrep™ gradient. Fractions were analyzed by Western blotting using antibodies against AHNAK, annexin 2, and S100A10. Caveolin-1 was used as a marker of lipid raft–containing fraction. (B) Confocal microscopy analysis of FITC-cholera toxin β chain (a) and AHNAK (b) colocalization in MDCK cells. (c) Merged image. (C) Specific release of membrane-bound AHNAK by sequestration of membrane cholesterol. Confocal microscopy analysis of AHNAK (a, b, e, and f) and annexin 2 (HH7; c, d, g, and h) in MCF-7 (a–d) and MDCK (e–h) cells not treated (a, c, e, and g) or incubated with 3.8 mM of methyl-β-cyclodextrin (MβCD) for 15 min (b and d) or 1 h (f and h) at 37°C before fixation.

Mentions: In addition to the Ca2+-dependent association of annexin 2 with phospholipids, annexin 2 can also associate with the plasma membrane lipid raft microdomains in a cholesterol-dependent manner (Oliferenko et al., 1999; Babiychuk and Draeger, 2000; Babiychuk et al., 2002). Several laboratories have reported that annexin 2 may link lipid rafts with the cortical cytoskeleton (Oliferenko et al., 1999), and that annexin 2 recruits signaling proteins to intercellular junctions in a cholesterol-dependent manner (Hansen et al., 2002). To test a possible association of AHNAK with the annexin 2/S100A10 complex within lipid rafts, we performed flotation experiments in OptiPrep™ gradients in the presence of cold Triton X-100 (Fig. 4 A). In this gradient, the detergent-insoluble lipid rafts will float at the interphase between the 0 and 20% OptiPrep™ layers. In MDCK cells, a significant amount of AHNAK partitioned with annexin 2 and S100A10 into the lipid raft fraction together with caveolin-1, a known cholesterol-binding protein (Fig. 4 A). Not all the annexin 2 cosedimented with AHNAK, suggesting that only a portion of the annexin 2/S100A10 complex interacts with AHNAK and associates with the lipid rafts. We confirmed the association of AHNAK with lipid rafts in whole cells by colocalization of AHNAK with the FITC-labeled β subunit of cholera toxin, which specifically binds the lipid raft ganglioside GM1 (Harder et al., 1998). Confocal laser scanning microscopy of MCF-7 cells showed an extensive overlap of AHNAK staining with the FITC-labeled cholera toxin β chain at the plasma membrane, including intercellular junctions (Fig. 4 B). Furthermore, depletion of plasma membrane cholesterol in MDCK and MCF-7 cells with methyl-β-cyclodextrin released a population of annexin 2 from the plasma membrane and totally abolished the junctional membrane localization of AHNAK, causing their redistribution to the cell cytoplasm (Fig. 4 C).


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 and annexin 2/S100A10 associate with cholesterol-rich membranes. (A) AHNAK and annexin 2/S100A10 cofractionate to the lipid raft fractions. MDCK cells were lysed in buffer containing 1% Triton X-100 on ice, and flotation fractionation was performed in a 5–40% OptiPrep™ gradient. Fractions were analyzed by Western blotting using antibodies against AHNAK, annexin 2, and S100A10. Caveolin-1 was used as a marker of lipid raft–containing fraction. (B) Confocal microscopy analysis of FITC-cholera toxin β chain (a) and AHNAK (b) colocalization in MDCK cells. (c) Merged image. (C) Specific release of membrane-bound AHNAK by sequestration of membrane cholesterol. Confocal microscopy analysis of AHNAK (a, b, e, and f) and annexin 2 (HH7; c, d, g, and h) in MCF-7 (a–d) and MDCK (e–h) cells not treated (a, c, e, and g) or incubated with 3.8 mM of methyl-β-cyclodextrin (MβCD) for 15 min (b and d) or 1 h (f and h) at 37°C before fixation.
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Related In: Results  -  Collection

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fig4: AHNAK and annexin 2/S100A10 associate with cholesterol-rich membranes. (A) AHNAK and annexin 2/S100A10 cofractionate to the lipid raft fractions. MDCK cells were lysed in buffer containing 1% Triton X-100 on ice, and flotation fractionation was performed in a 5–40% OptiPrep™ gradient. Fractions were analyzed by Western blotting using antibodies against AHNAK, annexin 2, and S100A10. Caveolin-1 was used as a marker of lipid raft–containing fraction. (B) Confocal microscopy analysis of FITC-cholera toxin β chain (a) and AHNAK (b) colocalization in MDCK cells. (c) Merged image. (C) Specific release of membrane-bound AHNAK by sequestration of membrane cholesterol. Confocal microscopy analysis of AHNAK (a, b, e, and f) and annexin 2 (HH7; c, d, g, and h) in MCF-7 (a–d) and MDCK (e–h) cells not treated (a, c, e, and g) or incubated with 3.8 mM of methyl-β-cyclodextrin (MβCD) for 15 min (b and d) or 1 h (f and h) at 37°C before fixation.
Mentions: In addition to the Ca2+-dependent association of annexin 2 with phospholipids, annexin 2 can also associate with the plasma membrane lipid raft microdomains in a cholesterol-dependent manner (Oliferenko et al., 1999; Babiychuk and Draeger, 2000; Babiychuk et al., 2002). Several laboratories have reported that annexin 2 may link lipid rafts with the cortical cytoskeleton (Oliferenko et al., 1999), and that annexin 2 recruits signaling proteins to intercellular junctions in a cholesterol-dependent manner (Hansen et al., 2002). To test a possible association of AHNAK with the annexin 2/S100A10 complex within lipid rafts, we performed flotation experiments in OptiPrep™ gradients in the presence of cold Triton X-100 (Fig. 4 A). In this gradient, the detergent-insoluble lipid rafts will float at the interphase between the 0 and 20% OptiPrep™ layers. In MDCK cells, a significant amount of AHNAK partitioned with annexin 2 and S100A10 into the lipid raft fraction together with caveolin-1, a known cholesterol-binding protein (Fig. 4 A). Not all the annexin 2 cosedimented with AHNAK, suggesting that only a portion of the annexin 2/S100A10 complex interacts with AHNAK and associates with the lipid rafts. We confirmed the association of AHNAK with lipid rafts in whole cells by colocalization of AHNAK with the FITC-labeled β subunit of cholera toxin, which specifically binds the lipid raft ganglioside GM1 (Harder et al., 1998). Confocal laser scanning microscopy of MCF-7 cells showed an extensive overlap of AHNAK staining with the FITC-labeled cholera toxin β chain at the plasma membrane, including intercellular junctions (Fig. 4 B). Furthermore, depletion of plasma membrane cholesterol in MDCK and MCF-7 cells with methyl-β-cyclodextrin released a population of annexin 2 from the plasma membrane and totally abolished the junctional membrane localization of AHNAK, causing their redistribution to the cell cytoplasm (Fig. 4 C).

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