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Integrin-specific signaling pathways controlling focal adhesion formation and cell migration.

Mostafavi-Pour Z, Askari JA, Parkinson SJ, Parker PJ, Ng TT, Humphries MJ - J. Cell Biol. (2003)

Bottom Line: After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties.First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not.Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
The fibronectin (FN)-binding integrins alpha4beta1 and alpha5beta1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not. Second, adhesion via alpha5beta1 caused an eightfold increase in protein kinase Calpha (PKCalpha) activation, but only basal PKCalpha activity was observed after adhesion via alpha4beta1. Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCalpha signaling as central to the functional differences between alpha4beta1 and alpha5beta1.

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Double immunofluorescence staining for vinculin and integrin β1 in A375-SM cells spread on different adhesive substrates. Cells were incubated for 2 h on 10 μg/ml of each substrate, with or without a further 30 min incubation with 5 μg/ml H/0 or 1:50 anti-syndecan-4. Heparin was added at 100 μg/ml simultaneously with H/0. Cells were fixed, permeabilized, and stained for vinculin (green) and integrin β1 (red). (A–C) Cells spread on 50K alone. The arrowhead in the merged image (C) indicates integrin+/vinculin− clusters. (D–F) Cells prespread on 50K for 2 h followed by addition of H/0. The arrowhead in F indicates colocalization of vinculin and integrin β1. (G–I) Cells prespread on 50K followed by treatment with H/0 in the presence of heparin. The arrowhead in I again indicates integrin+/vinculin− clusters. (J–L) Cells prespread on 50K followed by addition of anti-syndecan-4 antibody. The arrowhead in L indicates colocalization of vinculin and integrin β1. Cells spread on either H/120 (M–O) or H/120 treated with H/0 (P–R) exhibit colocalization of vinculin and integrin β1 (arrowheads in O and R). Addition of heparin and H/0 to cells prespread on H/120 (S–U) had no discernible effect. Bar, 20 μm.
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fig2: Double immunofluorescence staining for vinculin and integrin β1 in A375-SM cells spread on different adhesive substrates. Cells were incubated for 2 h on 10 μg/ml of each substrate, with or without a further 30 min incubation with 5 μg/ml H/0 or 1:50 anti-syndecan-4. Heparin was added at 100 μg/ml simultaneously with H/0. Cells were fixed, permeabilized, and stained for vinculin (green) and integrin β1 (red). (A–C) Cells spread on 50K alone. The arrowhead in the merged image (C) indicates integrin+/vinculin− clusters. (D–F) Cells prespread on 50K for 2 h followed by addition of H/0. The arrowhead in F indicates colocalization of vinculin and integrin β1. (G–I) Cells prespread on 50K followed by treatment with H/0 in the presence of heparin. The arrowhead in I again indicates integrin+/vinculin− clusters. (J–L) Cells prespread on 50K followed by addition of anti-syndecan-4 antibody. The arrowhead in L indicates colocalization of vinculin and integrin β1. Cells spread on either H/120 (M–O) or H/120 treated with H/0 (P–R) exhibit colocalization of vinculin and integrin β1 (arrowheads in O and R). Addition of heparin and H/0 to cells prespread on H/120 (S–U) had no discernible effect. Bar, 20 μm.

Mentions: To compare the role of syndecans in focal adhesion formation mediated by the integrins α4β1 and α5β1, A375-SM melanoma cells were plated on defined fragments of FN that interact specifically with each receptor (Fig. 1) . A375-SM cells have previously been shown to express α4β1 and α5β1 (Mould et al., 1990), and we have found by FACS analysis that they also express syndecan-4 (unpublished data). When cells were plated onto a recombinant 50K fragment spanning the CCBD of FN (recognized by α5β1), less than 15% of spread cells formed vinculin-containing focal adhesions (Table I a; Fig. 2 A). Although vinculin was rarely concentrated at sites of attachment, it was notable that these sites did contain α5 (unpublished data) and β1 integrins (Fig. 2 B), suggesting that integrin engagement and clustering had been dissociated from assembly of cytoskeletal structures. It is also notable that talin, α-actinin, focal adhesion kinase, and paxillin were absent from adhesion contacts (unpublished data). When cells were seeded on 50K and the medium was supplemented with a low concentration of soluble heparin-binding fragment of FN (5 μg/ml H/0), there was a significant increase in the percentage of spread cells (from 40–50% to 70–80%), and almost 90% of the spread cells now formed vinculin- and integrin-containing focal adhesions (Table I a; Fig. 2, D–F). The stimulatory effect of H/0 was abolished by co-incubation with heparin (Table I b; Fig. 2, G–I), and could be mimicked by addition of soluble anti-syndecan-4 mAb 5G9 (Fig. 2, J–L). These results confirm the synergistic link between integrin α5β1 and syndecan-4 for focal adhesion formation.


Integrin-specific signaling pathways controlling focal adhesion formation and cell migration.

Mostafavi-Pour Z, Askari JA, Parkinson SJ, Parker PJ, Ng TT, Humphries MJ - J. Cell Biol. (2003)

Double immunofluorescence staining for vinculin and integrin β1 in A375-SM cells spread on different adhesive substrates. Cells were incubated for 2 h on 10 μg/ml of each substrate, with or without a further 30 min incubation with 5 μg/ml H/0 or 1:50 anti-syndecan-4. Heparin was added at 100 μg/ml simultaneously with H/0. Cells were fixed, permeabilized, and stained for vinculin (green) and integrin β1 (red). (A–C) Cells spread on 50K alone. The arrowhead in the merged image (C) indicates integrin+/vinculin− clusters. (D–F) Cells prespread on 50K for 2 h followed by addition of H/0. The arrowhead in F indicates colocalization of vinculin and integrin β1. (G–I) Cells prespread on 50K followed by treatment with H/0 in the presence of heparin. The arrowhead in I again indicates integrin+/vinculin− clusters. (J–L) Cells prespread on 50K followed by addition of anti-syndecan-4 antibody. The arrowhead in L indicates colocalization of vinculin and integrin β1. Cells spread on either H/120 (M–O) or H/120 treated with H/0 (P–R) exhibit colocalization of vinculin and integrin β1 (arrowheads in O and R). Addition of heparin and H/0 to cells prespread on H/120 (S–U) had no discernible effect. Bar, 20 μm.
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Related In: Results  -  Collection

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fig2: Double immunofluorescence staining for vinculin and integrin β1 in A375-SM cells spread on different adhesive substrates. Cells were incubated for 2 h on 10 μg/ml of each substrate, with or without a further 30 min incubation with 5 μg/ml H/0 or 1:50 anti-syndecan-4. Heparin was added at 100 μg/ml simultaneously with H/0. Cells were fixed, permeabilized, and stained for vinculin (green) and integrin β1 (red). (A–C) Cells spread on 50K alone. The arrowhead in the merged image (C) indicates integrin+/vinculin− clusters. (D–F) Cells prespread on 50K for 2 h followed by addition of H/0. The arrowhead in F indicates colocalization of vinculin and integrin β1. (G–I) Cells prespread on 50K followed by treatment with H/0 in the presence of heparin. The arrowhead in I again indicates integrin+/vinculin− clusters. (J–L) Cells prespread on 50K followed by addition of anti-syndecan-4 antibody. The arrowhead in L indicates colocalization of vinculin and integrin β1. Cells spread on either H/120 (M–O) or H/120 treated with H/0 (P–R) exhibit colocalization of vinculin and integrin β1 (arrowheads in O and R). Addition of heparin and H/0 to cells prespread on H/120 (S–U) had no discernible effect. Bar, 20 μm.
Mentions: To compare the role of syndecans in focal adhesion formation mediated by the integrins α4β1 and α5β1, A375-SM melanoma cells were plated on defined fragments of FN that interact specifically with each receptor (Fig. 1) . A375-SM cells have previously been shown to express α4β1 and α5β1 (Mould et al., 1990), and we have found by FACS analysis that they also express syndecan-4 (unpublished data). When cells were plated onto a recombinant 50K fragment spanning the CCBD of FN (recognized by α5β1), less than 15% of spread cells formed vinculin-containing focal adhesions (Table I a; Fig. 2 A). Although vinculin was rarely concentrated at sites of attachment, it was notable that these sites did contain α5 (unpublished data) and β1 integrins (Fig. 2 B), suggesting that integrin engagement and clustering had been dissociated from assembly of cytoskeletal structures. It is also notable that talin, α-actinin, focal adhesion kinase, and paxillin were absent from adhesion contacts (unpublished data). When cells were seeded on 50K and the medium was supplemented with a low concentration of soluble heparin-binding fragment of FN (5 μg/ml H/0), there was a significant increase in the percentage of spread cells (from 40–50% to 70–80%), and almost 90% of the spread cells now formed vinculin- and integrin-containing focal adhesions (Table I a; Fig. 2, D–F). The stimulatory effect of H/0 was abolished by co-incubation with heparin (Table I b; Fig. 2, G–I), and could be mimicked by addition of soluble anti-syndecan-4 mAb 5G9 (Fig. 2, J–L). These results confirm the synergistic link between integrin α5β1 and syndecan-4 for focal adhesion formation.

Bottom Line: After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties.First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not.Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
The fibronectin (FN)-binding integrins alpha4beta1 and alpha5beta1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not. Second, adhesion via alpha5beta1 caused an eightfold increase in protein kinase Calpha (PKCalpha) activation, but only basal PKCalpha activity was observed after adhesion via alpha4beta1. Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCalpha signaling as central to the functional differences between alpha4beta1 and alpha5beta1.

Show MeSH
Related in: MedlinePlus