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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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PI(4,5)P2 and immobilized ligand are required for integrin clustering. (A and B) Stable β3-EGFP integrin–expressing B16F1 cells were cultured in neomycin sulfate–containing medium for 36 h. Control (A) or Mn2+-stimulated (B) cells were fixed and analyzed by confocal microscopy. The corresponding inset represents the IRM image of the boxed region of the cell in A and B, respectively. (C) Averaged histograms (n > 20) of Mn2+-induced integrin clustering in respect to increasing concentrations of neomycin sulfate. The histogram analysis of the inactive integrin mutant D119Y (Fig. 1 F) is shown as a negative reference. (D–F) Integrin clustering on a micropatterned substrate of vitronectin/fibronectin. Epifluorescence images of Alexa 568–labeled fibronectin (D) and corresponding β3-EGFP integrin fluorescence of B16F1 cells before Mn2+ addition (E). The same cells were imaged 10 min after treatment with Mn2+ (F). Note the exclusive accumulation of de novo integrin clusters on ECM-coated, but not noncoated, surfaces. (G–I) Inhibition of de novo integrin clustering in the presence of cRGD. Stable β3-EGFP integrin–expressing B16F1 cells were cultured overnight in complete medium and fixed 20 min after the addition of 10 μM cRGD (G), 0.5 mM Mn2+ (H), or both reagents (I). Confocal images of the integrin fluorescence at the level of the glass coverslip indicate the inhibition of Mn2+-induced integrin clustering by cRGD. Bars: (A,B, and G–I) 35 μm; (D–F) 50 μm.
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fig3: PI(4,5)P2 and immobilized ligand are required for integrin clustering. (A and B) Stable β3-EGFP integrin–expressing B16F1 cells were cultured in neomycin sulfate–containing medium for 36 h. Control (A) or Mn2+-stimulated (B) cells were fixed and analyzed by confocal microscopy. The corresponding inset represents the IRM image of the boxed region of the cell in A and B, respectively. (C) Averaged histograms (n > 20) of Mn2+-induced integrin clustering in respect to increasing concentrations of neomycin sulfate. The histogram analysis of the inactive integrin mutant D119Y (Fig. 1 F) is shown as a negative reference. (D–F) Integrin clustering on a micropatterned substrate of vitronectin/fibronectin. Epifluorescence images of Alexa 568–labeled fibronectin (D) and corresponding β3-EGFP integrin fluorescence of B16F1 cells before Mn2+ addition (E). The same cells were imaged 10 min after treatment with Mn2+ (F). Note the exclusive accumulation of de novo integrin clusters on ECM-coated, but not noncoated, surfaces. (G–I) Inhibition of de novo integrin clustering in the presence of cRGD. Stable β3-EGFP integrin–expressing B16F1 cells were cultured overnight in complete medium and fixed 20 min after the addition of 10 μM cRGD (G), 0.5 mM Mn2+ (H), or both reagents (I). Confocal images of the integrin fluorescence at the level of the glass coverslip indicate the inhibition of Mn2+-induced integrin clustering by cRGD. Bars: (A,B, and G–I) 35 μm; (D–F) 50 μm.

Mentions: It has been demonstrated that peptides, representing the cytoplasmic tail of integrins, bind to talin. In addition, phosphoinositol-4,5-bisphosphate (PI[4,5]P2) binding to talin induces a conformational change that facilitates the interaction of the talin head domain with the cytoplasmic tail of β3 integrins (Martel et al., 2001). Therefore, we asked whether PI(4,5)P2 is a critical cofactor in the clustering of high-affinity integrins. We treated β3 integrin–expressing cells with increasing doses of neomycin sulfate, a drug known to selectively bind and sequester PI(4,5)P2 (Arbuzova et al., 2000; Laux et al., 2000). The sequestration of PI(4,5)P2 by 10 mM neomycin dramatically reduced the formation of β3 integrin clusters in the periphery of control cells (Fig. 3 A), as well as Mn2+-induced, de novo integrin clusters underneath the main cell body (Fig. 3 B). Despite the reduction of integrin clustering under the main cell body, some remaining integrin clusters were found in the cell periphery often associated with filopodia. These thin, fingerlike integrin clusters were in contact with the substrate as suggested by the corresponding IRM image (Fig. 3, A and B, insets). Similar to Mn2+-stimulated WT cells, 10 mM neomycin suppressed peripheral focal contacts and integrin clusters under the main cell body in D723A and N305T mutant β3 integrin–transfected cells (unpublished data). To better characterize the effect of neomycin on integrin clustering, we performed dose response analysis with WT β3 integrin–expressing cells. Mn2+-induced integrin clustering was efficiently prevented at 1 mM neomycin sulfate (Fig. 3 C). To exclude the possibility that the neomycin sulfate–dependent inhibition of integrin clustering was indirectly caused by the drug's effect on the actin cytoskeleton (Laux et al., 2000; Kwik et al., 2003), we treated cells with cD before Mn2+ stimulation. Irrespective of the state of the actin cytoskeleton, neomycin sulfate prevented the formation of Mn2+-induced integrin clusters (unpublished data). To test whether PI(4,5)P2 was also important for the maintenance of integrin clusters, we tested whether its sequestration would affect previously clustered integrins. The addition of 10 mM neomycin sulfate to Mn2+-stimulated cells dispersed integrin clusters within 1 h (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200503017/DC1). Nevertheless, the dispersed but activated integrins were still able to link the cells to the substrate as revealed by IRM (unpublished data). These results demonstrate that PI(4,5)P2 is involved in the induction and stabilization of the lateral association of integrins, either by increasing the affinity of talin for integrins (Martel et al., 2001) or by oligomerization of the integrin–talin complex through interaction with lipid domains containing PI(4,5)P2.


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)

PI(4,5)P2 and immobilized ligand are required for integrin clustering. (A and B) Stable β3-EGFP integrin–expressing B16F1 cells were cultured in neomycin sulfate–containing medium for 36 h. Control (A) or Mn2+-stimulated (B) cells were fixed and analyzed by confocal microscopy. The corresponding inset represents the IRM image of the boxed region of the cell in A and B, respectively. (C) Averaged histograms (n > 20) of Mn2+-induced integrin clustering in respect to increasing concentrations of neomycin sulfate. The histogram analysis of the inactive integrin mutant D119Y (Fig. 1 F) is shown as a negative reference. (D–F) Integrin clustering on a micropatterned substrate of vitronectin/fibronectin. Epifluorescence images of Alexa 568–labeled fibronectin (D) and corresponding β3-EGFP integrin fluorescence of B16F1 cells before Mn2+ addition (E). The same cells were imaged 10 min after treatment with Mn2+ (F). Note the exclusive accumulation of de novo integrin clusters on ECM-coated, but not noncoated, surfaces. (G–I) Inhibition of de novo integrin clustering in the presence of cRGD. Stable β3-EGFP integrin–expressing B16F1 cells were cultured overnight in complete medium and fixed 20 min after the addition of 10 μM cRGD (G), 0.5 mM Mn2+ (H), or both reagents (I). Confocal images of the integrin fluorescence at the level of the glass coverslip indicate the inhibition of Mn2+-induced integrin clustering by cRGD. Bars: (A,B, and G–I) 35 μm; (D–F) 50 μm.
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fig3: PI(4,5)P2 and immobilized ligand are required for integrin clustering. (A and B) Stable β3-EGFP integrin–expressing B16F1 cells were cultured in neomycin sulfate–containing medium for 36 h. Control (A) or Mn2+-stimulated (B) cells were fixed and analyzed by confocal microscopy. The corresponding inset represents the IRM image of the boxed region of the cell in A and B, respectively. (C) Averaged histograms (n > 20) of Mn2+-induced integrin clustering in respect to increasing concentrations of neomycin sulfate. The histogram analysis of the inactive integrin mutant D119Y (Fig. 1 F) is shown as a negative reference. (D–F) Integrin clustering on a micropatterned substrate of vitronectin/fibronectin. Epifluorescence images of Alexa 568–labeled fibronectin (D) and corresponding β3-EGFP integrin fluorescence of B16F1 cells before Mn2+ addition (E). The same cells were imaged 10 min after treatment with Mn2+ (F). Note the exclusive accumulation of de novo integrin clusters on ECM-coated, but not noncoated, surfaces. (G–I) Inhibition of de novo integrin clustering in the presence of cRGD. Stable β3-EGFP integrin–expressing B16F1 cells were cultured overnight in complete medium and fixed 20 min after the addition of 10 μM cRGD (G), 0.5 mM Mn2+ (H), or both reagents (I). Confocal images of the integrin fluorescence at the level of the glass coverslip indicate the inhibition of Mn2+-induced integrin clustering by cRGD. Bars: (A,B, and G–I) 35 μm; (D–F) 50 μm.
Mentions: It has been demonstrated that peptides, representing the cytoplasmic tail of integrins, bind to talin. In addition, phosphoinositol-4,5-bisphosphate (PI[4,5]P2) binding to talin induces a conformational change that facilitates the interaction of the talin head domain with the cytoplasmic tail of β3 integrins (Martel et al., 2001). Therefore, we asked whether PI(4,5)P2 is a critical cofactor in the clustering of high-affinity integrins. We treated β3 integrin–expressing cells with increasing doses of neomycin sulfate, a drug known to selectively bind and sequester PI(4,5)P2 (Arbuzova et al., 2000; Laux et al., 2000). The sequestration of PI(4,5)P2 by 10 mM neomycin dramatically reduced the formation of β3 integrin clusters in the periphery of control cells (Fig. 3 A), as well as Mn2+-induced, de novo integrin clusters underneath the main cell body (Fig. 3 B). Despite the reduction of integrin clustering under the main cell body, some remaining integrin clusters were found in the cell periphery often associated with filopodia. These thin, fingerlike integrin clusters were in contact with the substrate as suggested by the corresponding IRM image (Fig. 3, A and B, insets). Similar to Mn2+-stimulated WT cells, 10 mM neomycin suppressed peripheral focal contacts and integrin clusters under the main cell body in D723A and N305T mutant β3 integrin–transfected cells (unpublished data). To better characterize the effect of neomycin on integrin clustering, we performed dose response analysis with WT β3 integrin–expressing cells. Mn2+-induced integrin clustering was efficiently prevented at 1 mM neomycin sulfate (Fig. 3 C). To exclude the possibility that the neomycin sulfate–dependent inhibition of integrin clustering was indirectly caused by the drug's effect on the actin cytoskeleton (Laux et al., 2000; Kwik et al., 2003), we treated cells with cD before Mn2+ stimulation. Irrespective of the state of the actin cytoskeleton, neomycin sulfate prevented the formation of Mn2+-induced integrin clusters (unpublished data). To test whether PI(4,5)P2 was also important for the maintenance of integrin clusters, we tested whether its sequestration would affect previously clustered integrins. The addition of 10 mM neomycin sulfate to Mn2+-stimulated cells dispersed integrin clusters within 1 h (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200503017/DC1). Nevertheless, the dispersed but activated integrins were still able to link the cells to the substrate as revealed by IRM (unpublished data). These results demonstrate that PI(4,5)P2 is involved in the induction and stabilization of the lateral association of integrins, either by increasing the affinity of talin for integrins (Martel et al., 2001) or by oligomerization of the integrin–talin complex through interaction with lipid domains containing PI(4,5)P2.

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