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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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The head domain of talin induces β3 integrin clustering. Cells stably expressing β3-EGFP integrin were transiently transfected with enhanced cyan fluorescent protein (ECFP)-tagged human NH2-terminal talin head domain and cultured in complete medium. 48 h after transfection, cells were fixed and observed by confocal microscopy. Talin head domain transfected control (A and B) or Mn2+-treated cells (20 min; 0.5 mM final concentration; D and E) revealed extensive integrin clustering (A and D). (C) Histogram analysis of integrin clustering in talin head transfected cells with or without the addition of Mn2+. (F) Analysis of the relative cell surface occupied by β3-EGFP integrin clusters in cells transfected with ECFP-talin head domain or empty ECFP vector and treated with or without Mn2+. Error bars represent the standard deviation of at least 20 double-transfected cells. (G and H) Confocal images of β3-EGFP integrin and ECFP-talin head domain in a Mn2+-stimulated, weakly talin-expressing cell. Note the similar staining pattern between integrins and the talin head domain (G' and H'). Data for graphs in C and F are from one out of three similar experiments. Bar, 27 μm.
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fig4: The head domain of talin induces β3 integrin clustering. Cells stably expressing β3-EGFP integrin were transiently transfected with enhanced cyan fluorescent protein (ECFP)-tagged human NH2-terminal talin head domain and cultured in complete medium. 48 h after transfection, cells were fixed and observed by confocal microscopy. Talin head domain transfected control (A and B) or Mn2+-treated cells (20 min; 0.5 mM final concentration; D and E) revealed extensive integrin clustering (A and D). (C) Histogram analysis of integrin clustering in talin head transfected cells with or without the addition of Mn2+. (F) Analysis of the relative cell surface occupied by β3-EGFP integrin clusters in cells transfected with ECFP-talin head domain or empty ECFP vector and treated with or without Mn2+. Error bars represent the standard deviation of at least 20 double-transfected cells. (G and H) Confocal images of β3-EGFP integrin and ECFP-talin head domain in a Mn2+-stimulated, weakly talin-expressing cell. Note the similar staining pattern between integrins and the talin head domain (G' and H'). Data for graphs in C and F are from one out of three similar experiments. Bar, 27 μm.

Mentions: As demonstrated in Fig. 2, the specific recruitment of talin to Mn2+-induced integrin clusters suggests that talin is critically involved in the formation of integrin clusters. Moreover, because talin exists as an anti-parallel dimer with the integrin-binding head domains positioned at both ends (Isenberg and Goldmann, 1998), talin represents a bona fide intracellular integrin cross-linker. To test whether the dimeric form of talin is required for integrin clustering, we overexpressed the monomeric integrin binding head domain of talin (residues 1–435; Yan et al., 2001) as a CFP-tagged chimera in stable β3-GFP integrin–expressing cells, with the intention to dominantly suppress integrin clustering. Instead, the overexpression of the isolated head domain of talin induced an increase in integrin clustering (Fig. 4, A–C). Similarly, Mn2+ stimulation of cells that overexpressed the head domain of talin induced a greater increase in integrin clustering, covering more than half of the cell surface (Fig. 4, D and E). In most cells, the cytoplasmic expression of talin was very high, making it impossible to determine whether the talin head domain only activated integrins or was also involved in their clustering. However, after Mn2+ stimulation, high levels of the talin head domain were no longer required for integrin activation, which nevertheless resulted in efficient clustering of integrin at low talin expression levels (Fig. 4, G and H). In these clusters (Fig. 4 G), we detected a colocalization with the head domain of talin (Fig. 4 H). These data suggest that the monomeric talin head domain possesses the ability to induce integrin activation and clustering. Because talin-dependent integrin clustering is sensitive to neomycin treatment (unpublished data), integrin clustering may be induced by the binding of the talin head domain to multivalent PI(4,5)P2 lipid domains.


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)

The head domain of talin induces β3 integrin clustering. Cells stably expressing β3-EGFP integrin were transiently transfected with enhanced cyan fluorescent protein (ECFP)-tagged human NH2-terminal talin head domain and cultured in complete medium. 48 h after transfection, cells were fixed and observed by confocal microscopy. Talin head domain transfected control (A and B) or Mn2+-treated cells (20 min; 0.5 mM final concentration; D and E) revealed extensive integrin clustering (A and D). (C) Histogram analysis of integrin clustering in talin head transfected cells with or without the addition of Mn2+. (F) Analysis of the relative cell surface occupied by β3-EGFP integrin clusters in cells transfected with ECFP-talin head domain or empty ECFP vector and treated with or without Mn2+. Error bars represent the standard deviation of at least 20 double-transfected cells. (G and H) Confocal images of β3-EGFP integrin and ECFP-talin head domain in a Mn2+-stimulated, weakly talin-expressing cell. Note the similar staining pattern between integrins and the talin head domain (G' and H'). Data for graphs in C and F are from one out of three similar experiments. Bar, 27 μm.
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fig4: The head domain of talin induces β3 integrin clustering. Cells stably expressing β3-EGFP integrin were transiently transfected with enhanced cyan fluorescent protein (ECFP)-tagged human NH2-terminal talin head domain and cultured in complete medium. 48 h after transfection, cells were fixed and observed by confocal microscopy. Talin head domain transfected control (A and B) or Mn2+-treated cells (20 min; 0.5 mM final concentration; D and E) revealed extensive integrin clustering (A and D). (C) Histogram analysis of integrin clustering in talin head transfected cells with or without the addition of Mn2+. (F) Analysis of the relative cell surface occupied by β3-EGFP integrin clusters in cells transfected with ECFP-talin head domain or empty ECFP vector and treated with or without Mn2+. Error bars represent the standard deviation of at least 20 double-transfected cells. (G and H) Confocal images of β3-EGFP integrin and ECFP-talin head domain in a Mn2+-stimulated, weakly talin-expressing cell. Note the similar staining pattern between integrins and the talin head domain (G' and H'). Data for graphs in C and F are from one out of three similar experiments. Bar, 27 μm.
Mentions: As demonstrated in Fig. 2, the specific recruitment of talin to Mn2+-induced integrin clusters suggests that talin is critically involved in the formation of integrin clusters. Moreover, because talin exists as an anti-parallel dimer with the integrin-binding head domains positioned at both ends (Isenberg and Goldmann, 1998), talin represents a bona fide intracellular integrin cross-linker. To test whether the dimeric form of talin is required for integrin clustering, we overexpressed the monomeric integrin binding head domain of talin (residues 1–435; Yan et al., 2001) as a CFP-tagged chimera in stable β3-GFP integrin–expressing cells, with the intention to dominantly suppress integrin clustering. Instead, the overexpression of the isolated head domain of talin induced an increase in integrin clustering (Fig. 4, A–C). Similarly, Mn2+ stimulation of cells that overexpressed the head domain of talin induced a greater increase in integrin clustering, covering more than half of the cell surface (Fig. 4, D and E). In most cells, the cytoplasmic expression of talin was very high, making it impossible to determine whether the talin head domain only activated integrins or was also involved in their clustering. However, after Mn2+ stimulation, high levels of the talin head domain were no longer required for integrin activation, which nevertheless resulted in efficient clustering of integrin at low talin expression levels (Fig. 4, G and H). In these clusters (Fig. 4 G), we detected a colocalization with the head domain of talin (Fig. 4 H). These data suggest that the monomeric talin head domain possesses the ability to induce integrin activation and clustering. Because talin-dependent integrin clustering is sensitive to neomycin treatment (unpublished data), integrin clustering may be induced by the binding of the talin head domain to multivalent PI(4,5)P2 lipid domains.

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