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The mechanisms and dynamics of (alpha)v(beta)3 integrin clustering in living cells.

Cluzel C, Saltel F, Lussi J, Paulhe F, Imhof BA, Wehrle-Haller B - J. Cell Biol. (2005)

Bottom Line: Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2).Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2.The dynamic remodeling of this ternary complex controls cell motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunlogy, Centre Medical Universitaire, 1211 Geneva 4, Switzerland.

ABSTRACT
During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated alphavbeta3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin-independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn(2+)-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility.

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F-actin–independent integrin clustering and talin recruitment. Epifluorescence of fixed B16F1 and CS-1 cells exhibiting de novo formed β3 integrin clusters. Integrin fluorescence (A and B) and phalloidin stained F-actin (A' and B') in 0.5 mM of Mn2+-treated cells (20 min; A and A') or 10 μg/ml of cD/Mn2+-treated B16F1 cells (25 min of cD, followed by 20 min of cD/Mn2+; B and B'). Immunohistochemical analysis of focal adhesion adaptor proteins recruited to Mn2+-induced clusters of β3-EGFP integrins in B16F1 cells (C–G) and nontagged β3 integrins in CS-1 cells (H and I). Pairs of images show the distribution of the EGFP integrin fluorescence (C–G) or anti-β3 staining (H and I) and the respective immunohistochemical localization of talin (C' and H'), vinculin (D' and I'), paxillin (E'), phosphotyrosine (F'), and FAK (G'). Corresponding magnified views of the boxed areas in A–H and A'–H' are shown below each image pair (a–h and a'–h'). Bar, 25 μm.
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fig2: F-actin–independent integrin clustering and talin recruitment. Epifluorescence of fixed B16F1 and CS-1 cells exhibiting de novo formed β3 integrin clusters. Integrin fluorescence (A and B) and phalloidin stained F-actin (A' and B') in 0.5 mM of Mn2+-treated cells (20 min; A and A') or 10 μg/ml of cD/Mn2+-treated B16F1 cells (25 min of cD, followed by 20 min of cD/Mn2+; B and B'). Immunohistochemical analysis of focal adhesion adaptor proteins recruited to Mn2+-induced clusters of β3-EGFP integrins in B16F1 cells (C–G) and nontagged β3 integrins in CS-1 cells (H and I). Pairs of images show the distribution of the EGFP integrin fluorescence (C–G) or anti-β3 staining (H and I) and the respective immunohistochemical localization of talin (C' and H'), vinculin (D' and I'), paxillin (E'), phosphotyrosine (F'), and FAK (G'). Corresponding magnified views of the boxed areas in A–H and A'–H' are shown below each image pair (a–h and a'–h'). Bar, 25 μm.

Mentions: Clusters of Mn2+ or mutational activated integrins were found underneath the main cell body, a cellular localization rarely populated by focal contacts. Because integrins clustered within focal adhesions are mechanically linked to the actin cytoskeleton, we analyzed whether actin and/or focal adhesion adaptor proteins are recruited to de novo–formed integrin clusters. We analyzed whether phalloidin reactive F-actin was localized to Mn2+-induced integrin clusters. Mn2+ activation resulted in the formation of integrin clusters in cellular regions devoid of F-actin (Fig. 2, A and A', inset). De novo formation of integrin clustering independent of F-actin was confirmed by treatment of spread cells with cytochalasin D (cD) before Mn2+ activation. Despite the destruction of the actin cytoskeleton by cD, Mn2+-activated β3 integrins formed clusters (Fig. 2, B and B'). Because actin fibers were dispensable for integrin clustering, we asked whether adaptor proteins were recruited to clusters of activated integrins. Immunofluorescence staining with antitalin antibodies revealed an overlap with all clustered EGFP integrins in Mn2+-treated cells (Fig. 2, C and C'). This confirms the data that talin–integrin association is important for the formation of focal adhesions (Priddle et al., 1998). In contrast to talin, the focal adhesion adaptor proteins vinculin, paxillin, and FAK, as well as antiphosphotyrosine antibodies, did not associate with de novo–formed clusters of activated integrins (Fig. 2, D–G). An identical result was obtained in β3 integrin negative CS-1 hamster melanoma cells that had been transfected with non-EGFP–tagged β3 integrins (Fig. 2, H and I). This suggests that the observed clustering of activated integrins and selective talin recruitment is not influenced by the EGFP tag.


The mechanisms and dynamics of (alpha)v(beta)3 integrin clustering in living cells.

Cluzel C, Saltel F, Lussi J, Paulhe F, Imhof BA, Wehrle-Haller B - J. Cell Biol. (2005)

F-actin–independent integrin clustering and talin recruitment. Epifluorescence of fixed B16F1 and CS-1 cells exhibiting de novo formed β3 integrin clusters. Integrin fluorescence (A and B) and phalloidin stained F-actin (A' and B') in 0.5 mM of Mn2+-treated cells (20 min; A and A') or 10 μg/ml of cD/Mn2+-treated B16F1 cells (25 min of cD, followed by 20 min of cD/Mn2+; B and B'). Immunohistochemical analysis of focal adhesion adaptor proteins recruited to Mn2+-induced clusters of β3-EGFP integrins in B16F1 cells (C–G) and nontagged β3 integrins in CS-1 cells (H and I). Pairs of images show the distribution of the EGFP integrin fluorescence (C–G) or anti-β3 staining (H and I) and the respective immunohistochemical localization of talin (C' and H'), vinculin (D' and I'), paxillin (E'), phosphotyrosine (F'), and FAK (G'). Corresponding magnified views of the boxed areas in A–H and A'–H' are shown below each image pair (a–h and a'–h'). Bar, 25 μm.
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fig2: F-actin–independent integrin clustering and talin recruitment. Epifluorescence of fixed B16F1 and CS-1 cells exhibiting de novo formed β3 integrin clusters. Integrin fluorescence (A and B) and phalloidin stained F-actin (A' and B') in 0.5 mM of Mn2+-treated cells (20 min; A and A') or 10 μg/ml of cD/Mn2+-treated B16F1 cells (25 min of cD, followed by 20 min of cD/Mn2+; B and B'). Immunohistochemical analysis of focal adhesion adaptor proteins recruited to Mn2+-induced clusters of β3-EGFP integrins in B16F1 cells (C–G) and nontagged β3 integrins in CS-1 cells (H and I). Pairs of images show the distribution of the EGFP integrin fluorescence (C–G) or anti-β3 staining (H and I) and the respective immunohistochemical localization of talin (C' and H'), vinculin (D' and I'), paxillin (E'), phosphotyrosine (F'), and FAK (G'). Corresponding magnified views of the boxed areas in A–H and A'–H' are shown below each image pair (a–h and a'–h'). Bar, 25 μm.
Mentions: Clusters of Mn2+ or mutational activated integrins were found underneath the main cell body, a cellular localization rarely populated by focal contacts. Because integrins clustered within focal adhesions are mechanically linked to the actin cytoskeleton, we analyzed whether actin and/or focal adhesion adaptor proteins are recruited to de novo–formed integrin clusters. We analyzed whether phalloidin reactive F-actin was localized to Mn2+-induced integrin clusters. Mn2+ activation resulted in the formation of integrin clusters in cellular regions devoid of F-actin (Fig. 2, A and A', inset). De novo formation of integrin clustering independent of F-actin was confirmed by treatment of spread cells with cytochalasin D (cD) before Mn2+ activation. Despite the destruction of the actin cytoskeleton by cD, Mn2+-activated β3 integrins formed clusters (Fig. 2, B and B'). Because actin fibers were dispensable for integrin clustering, we asked whether adaptor proteins were recruited to clusters of activated integrins. Immunofluorescence staining with antitalin antibodies revealed an overlap with all clustered EGFP integrins in Mn2+-treated cells (Fig. 2, C and C'). This confirms the data that talin–integrin association is important for the formation of focal adhesions (Priddle et al., 1998). In contrast to talin, the focal adhesion adaptor proteins vinculin, paxillin, and FAK, as well as antiphosphotyrosine antibodies, did not associate with de novo–formed clusters of activated integrins (Fig. 2, D–G). An identical result was obtained in β3 integrin negative CS-1 hamster melanoma cells that had been transfected with non-EGFP–tagged β3 integrins (Fig. 2, H and I). This suggests that the observed clustering of activated integrins and selective talin recruitment is not influenced by the EGFP tag.

Bottom Line: Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2).Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2.The dynamic remodeling of this ternary complex controls cell motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunlogy, Centre Medical Universitaire, 1211 Geneva 4, Switzerland.

ABSTRACT
During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated alphavbeta3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin-independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn(2+)-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility.

Show MeSH
Related in: MedlinePlus