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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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Increased clustering of high-affinity integrins. Confocal images at the level of the ventral cell surface of mouse B16F1 melanoma cells stably transfected with WT (A and B), D723A (C), N305T (D), or D119Y (E) mutant β3-EGFP integrin. Cells were grown in control medium (A and C–E) or stimulated for 20 min in 0.5 mM of Mn2+-containing medium (B). Note the increased number of integrin clusters at the cell surfaces underlying the main cell body in B–D. (F) Quantification of the relative amount of integrin clustering by histogram analysis. Averaged (n > 20) cumulative histograms from cells as shown in A–E (Fig. S1). The vertical line represents the fluorescence threshold corresponding to 99% of the histogram area of the inactive integrin D119Y mutant. Confocal (G–I) and corresponding interference reflection images (J–L) of Mn2+-induced integrin clustering in a B16F1 cell cultured overnight in serum-containing medium. (G and J) The cell before Mn2+ addition; (H and K) the cell after 3 min of Mn2+ addition; (I and L) the cell after 9 min of Mn2+ addition. Magnified views of the area underneath the main cell body representing de novo–formed dot- and streaklike integrin clusters are represented in insets G′–L′. Bars: (A–E) 25 μm; (G–L) 37.5 μm.
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fig1: Increased clustering of high-affinity integrins. Confocal images at the level of the ventral cell surface of mouse B16F1 melanoma cells stably transfected with WT (A and B), D723A (C), N305T (D), or D119Y (E) mutant β3-EGFP integrin. Cells were grown in control medium (A and C–E) or stimulated for 20 min in 0.5 mM of Mn2+-containing medium (B). Note the increased number of integrin clusters at the cell surfaces underlying the main cell body in B–D. (F) Quantification of the relative amount of integrin clustering by histogram analysis. Averaged (n > 20) cumulative histograms from cells as shown in A–E (Fig. S1). The vertical line represents the fluorescence threshold corresponding to 99% of the histogram area of the inactive integrin D119Y mutant. Confocal (G–I) and corresponding interference reflection images (J–L) of Mn2+-induced integrin clustering in a B16F1 cell cultured overnight in serum-containing medium. (G and J) The cell before Mn2+ addition; (H and K) the cell after 3 min of Mn2+ addition; (I and L) the cell after 9 min of Mn2+ addition. Magnified views of the area underneath the main cell body representing de novo–formed dot- and streaklike integrin clusters are represented in insets G′–L′. Bars: (A–E) 25 μm; (G–L) 37.5 μm.

Mentions: In adherent cells, WT β3-EGFP integrins formed brightly fluorescent clusters at the cell periphery, representing focal contacts (Ballestrem et al., 2001; Fig. 1 A). In addition, nonclustered integrins were found evenly dispersed within the plasma membrane (Fig. 1 A). Because the population of cell surface integrins is in equilibrium between the activated and nonactivated forms, it is not clear to what extent activated integrins are present in the clustered or nonclustered state. To shift the equilibrium toward activated states of integrins, we treated cells with Mn2+. Only minutes after Mn2+ activation, we observed the formation of numerous irregularly shaped integrin clusters underneath the main cell body, in addition to the preexisting clusters of integrins at the periphery of the cell (Fig. 1 B). Similar to Mn2+-activated WT β3 integrin, the activated β3 integrin mutants D723A and N305T formed clusters underneath the entire cell body and in the periphery of the cell (Fig. 1, C and D). In contrast, the inactive β3 integrin mutant (D119Y), lacking the ability to bind ligand, was found evenly dispersed at the cell surface, exhibiting no apparent integrin clustering (Fig. 1 E). These results demonstrate that integrin activation correlates with integrin clustering and suggest a role for ligand binding in this process (Miyamoto et al., 1995).


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)

Increased clustering of high-affinity integrins. Confocal images at the level of the ventral cell surface of mouse B16F1 melanoma cells stably transfected with WT (A and B), D723A (C), N305T (D), or D119Y (E) mutant β3-EGFP integrin. Cells were grown in control medium (A and C–E) or stimulated for 20 min in 0.5 mM of Mn2+-containing medium (B). Note the increased number of integrin clusters at the cell surfaces underlying the main cell body in B–D. (F) Quantification of the relative amount of integrin clustering by histogram analysis. Averaged (n > 20) cumulative histograms from cells as shown in A–E (Fig. S1). The vertical line represents the fluorescence threshold corresponding to 99% of the histogram area of the inactive integrin D119Y mutant. Confocal (G–I) and corresponding interference reflection images (J–L) of Mn2+-induced integrin clustering in a B16F1 cell cultured overnight in serum-containing medium. (G and J) The cell before Mn2+ addition; (H and K) the cell after 3 min of Mn2+ addition; (I and L) the cell after 9 min of Mn2+ addition. Magnified views of the area underneath the main cell body representing de novo–formed dot- and streaklike integrin clusters are represented in insets G′–L′. Bars: (A–E) 25 μm; (G–L) 37.5 μm.
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fig1: Increased clustering of high-affinity integrins. Confocal images at the level of the ventral cell surface of mouse B16F1 melanoma cells stably transfected with WT (A and B), D723A (C), N305T (D), or D119Y (E) mutant β3-EGFP integrin. Cells were grown in control medium (A and C–E) or stimulated for 20 min in 0.5 mM of Mn2+-containing medium (B). Note the increased number of integrin clusters at the cell surfaces underlying the main cell body in B–D. (F) Quantification of the relative amount of integrin clustering by histogram analysis. Averaged (n > 20) cumulative histograms from cells as shown in A–E (Fig. S1). The vertical line represents the fluorescence threshold corresponding to 99% of the histogram area of the inactive integrin D119Y mutant. Confocal (G–I) and corresponding interference reflection images (J–L) of Mn2+-induced integrin clustering in a B16F1 cell cultured overnight in serum-containing medium. (G and J) The cell before Mn2+ addition; (H and K) the cell after 3 min of Mn2+ addition; (I and L) the cell after 9 min of Mn2+ addition. Magnified views of the area underneath the main cell body representing de novo–formed dot- and streaklike integrin clusters are represented in insets G′–L′. Bars: (A–E) 25 μm; (G–L) 37.5 μm.
Mentions: In adherent cells, WT β3-EGFP integrins formed brightly fluorescent clusters at the cell periphery, representing focal contacts (Ballestrem et al., 2001; Fig. 1 A). In addition, nonclustered integrins were found evenly dispersed within the plasma membrane (Fig. 1 A). Because the population of cell surface integrins is in equilibrium between the activated and nonactivated forms, it is not clear to what extent activated integrins are present in the clustered or nonclustered state. To shift the equilibrium toward activated states of integrins, we treated cells with Mn2+. Only minutes after Mn2+ activation, we observed the formation of numerous irregularly shaped integrin clusters underneath the main cell body, in addition to the preexisting clusters of integrins at the periphery of the cell (Fig. 1 B). Similar to Mn2+-activated WT β3 integrin, the activated β3 integrin mutants D723A and N305T formed clusters underneath the entire cell body and in the periphery of the cell (Fig. 1, C and D). In contrast, the inactive β3 integrin mutant (D119Y), lacking the ability to bind ligand, was found evenly dispersed at the cell surface, exhibiting no apparent integrin clustering (Fig. 1 E). These results demonstrate that integrin activation correlates with integrin clustering and suggest a role for ligand binding in this process (Miyamoto et al., 1995).

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