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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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Model of αvβ3 integrin activation and clustering. (A) Schematic view of a sequence of integrin activation and clustering. Talin is recruited and activated by membrane exposed PI(4,5)P2 lipids. Binding of the talin–PI(4,5)P2 complex to the cytoplasmic domain of integrin induces its “inside-out” activation (unfolding) allowing it to bind to immobilized extracellular ligands. Alternatively, integrins can interact with its extracellular ligands leading to “outside-in” activation and subsequent recruitment of PI(4,5)P2-bound talin, resulting in the formation of an “integrin preadhesion complex.” Subsequent clustering of this ternary complex is mediated by multivalent PI(4,5)P2-containing lipid domains. (B) Alternative modes of integrin activation, such as by the head domain of talin in response to calpain-mediated proteolytic cleavage or by low concentrations of soluble ligand (Legler et al., 2001), Mn2+, and activating mutations (N305T and D723A). These activation pathways can be modulated by the sequestration (Laux et al., 2000), synthesis, or degradation of PI(4,5)P2 lipids or by high doses of soluble ligands.
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fig6: Model of αvβ3 integrin activation and clustering. (A) Schematic view of a sequence of integrin activation and clustering. Talin is recruited and activated by membrane exposed PI(4,5)P2 lipids. Binding of the talin–PI(4,5)P2 complex to the cytoplasmic domain of integrin induces its “inside-out” activation (unfolding) allowing it to bind to immobilized extracellular ligands. Alternatively, integrins can interact with its extracellular ligands leading to “outside-in” activation and subsequent recruitment of PI(4,5)P2-bound talin, resulting in the formation of an “integrin preadhesion complex.” Subsequent clustering of this ternary complex is mediated by multivalent PI(4,5)P2-containing lipid domains. (B) Alternative modes of integrin activation, such as by the head domain of talin in response to calpain-mediated proteolytic cleavage or by low concentrations of soluble ligand (Legler et al., 2001), Mn2+, and activating mutations (N305T and D723A). These activation pathways can be modulated by the sequestration (Laux et al., 2000), synthesis, or degradation of PI(4,5)P2 lipids or by high doses of soluble ligands.

Mentions: The nature of the driving force that induces the lateral association of activated integrins is a matter of debate. One possibility put forward by Li et al. (2003) is that homophilic interactions between the transmembrane domains of activated integrins drive lateral clustering. This model implies that integrin clustering can occur independently of other integrin-binding proteins as long as the transmembrane domains of the α and β integrin subunit are physically separated from each other (e.g., separation induced by integrin activation). Other results, however, do not support the view that activated integrins can cluster spontaneously. Electron microscopic images of purified, activated αIIbβ3 integrins that are incorporated into lipid vesicles or planar membranes give no evidence for spontaneous clustering of activated integrins (Erb et al., 1997). In addition, Mn2+-activated αvβ3 integrins do not cluster spontaneously when exposed to a laminin-1 substrate to which they are unable to bind (unpublished data). Moreover, the disulfide bond scanning of the exofacial portions of the integrin αIIb and β3 transmembrane domains did not reveal a specific interaction of these domains after integrin activation in living cells (Luo et al., 2004). We now demonstrate that activated αvβ3 integrins require immobilized substrate, PI(4,5)P2 lipids, and the focal adhesion adaptor protein talin for clustering. Our data suggest that integrin clustering is controlled by a simple association–dissociation reaction that is influenced by the density of activated integrins in the plasma membrane. However, the equilibrium of this reaction can be shifted depending on the availability of immobilized substrate, PI(4,5)P2, and talin (Fig. 6 A).


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)

Model of αvβ3 integrin activation and clustering. (A) Schematic view of a sequence of integrin activation and clustering. Talin is recruited and activated by membrane exposed PI(4,5)P2 lipids. Binding of the talin–PI(4,5)P2 complex to the cytoplasmic domain of integrin induces its “inside-out” activation (unfolding) allowing it to bind to immobilized extracellular ligands. Alternatively, integrins can interact with its extracellular ligands leading to “outside-in” activation and subsequent recruitment of PI(4,5)P2-bound talin, resulting in the formation of an “integrin preadhesion complex.” Subsequent clustering of this ternary complex is mediated by multivalent PI(4,5)P2-containing lipid domains. (B) Alternative modes of integrin activation, such as by the head domain of talin in response to calpain-mediated proteolytic cleavage or by low concentrations of soluble ligand (Legler et al., 2001), Mn2+, and activating mutations (N305T and D723A). These activation pathways can be modulated by the sequestration (Laux et al., 2000), synthesis, or degradation of PI(4,5)P2 lipids or by high doses of soluble ligands.
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fig6: Model of αvβ3 integrin activation and clustering. (A) Schematic view of a sequence of integrin activation and clustering. Talin is recruited and activated by membrane exposed PI(4,5)P2 lipids. Binding of the talin–PI(4,5)P2 complex to the cytoplasmic domain of integrin induces its “inside-out” activation (unfolding) allowing it to bind to immobilized extracellular ligands. Alternatively, integrins can interact with its extracellular ligands leading to “outside-in” activation and subsequent recruitment of PI(4,5)P2-bound talin, resulting in the formation of an “integrin preadhesion complex.” Subsequent clustering of this ternary complex is mediated by multivalent PI(4,5)P2-containing lipid domains. (B) Alternative modes of integrin activation, such as by the head domain of talin in response to calpain-mediated proteolytic cleavage or by low concentrations of soluble ligand (Legler et al., 2001), Mn2+, and activating mutations (N305T and D723A). These activation pathways can be modulated by the sequestration (Laux et al., 2000), synthesis, or degradation of PI(4,5)P2 lipids or by high doses of soluble ligands.
Mentions: The nature of the driving force that induces the lateral association of activated integrins is a matter of debate. One possibility put forward by Li et al. (2003) is that homophilic interactions between the transmembrane domains of activated integrins drive lateral clustering. This model implies that integrin clustering can occur independently of other integrin-binding proteins as long as the transmembrane domains of the α and β integrin subunit are physically separated from each other (e.g., separation induced by integrin activation). Other results, however, do not support the view that activated integrins can cluster spontaneously. Electron microscopic images of purified, activated αIIbβ3 integrins that are incorporated into lipid vesicles or planar membranes give no evidence for spontaneous clustering of activated integrins (Erb et al., 1997). In addition, Mn2+-activated αvβ3 integrins do not cluster spontaneously when exposed to a laminin-1 substrate to which they are unable to bind (unpublished data). Moreover, the disulfide bond scanning of the exofacial portions of the integrin αIIb and β3 transmembrane domains did not reveal a specific interaction of these domains after integrin activation in living cells (Luo et al., 2004). We now demonstrate that activated αvβ3 integrins require immobilized substrate, PI(4,5)P2 lipids, and the focal adhesion adaptor protein talin for clustering. Our data suggest that integrin clustering is controlled by a simple association–dissociation reaction that is influenced by the density of activated integrins in the plasma membrane. However, the equilibrium of this reaction can be shifted depending on the availability of immobilized substrate, PI(4,5)P2, and talin (Fig. 6 A).

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