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Integrins control motile strategy through a Rho-cofilin pathway.

Danen EH, van Rheenen J, Franken W, Huveneers S, Sonneveld P, Jalink K, Sonnenberg A - J. Cell Biol. (2005)

Bottom Line: During wound healing, angiogenesis, and tumor invasion, cells often change their expression profiles of fibronectin-binding integrins.The activity of the small GTPase RhoA is particularly high in cells adhering by alpha5beta1, and inhibition of Rho signaling causes a switch from a beta1- to a beta3-associated mode of migration, whereas increased Rho activity has the opposite effect.Thus, alterations in integrin expression profiles allow cells to modulate several critical aspects of the motile machinery through Rho GTPases.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. e.danen@nki.nl

ABSTRACT
During wound healing, angiogenesis, and tumor invasion, cells often change their expression profiles of fibronectin-binding integrins. Here, we show that beta1 integrins promote random migration, whereas beta3 integrins promote persistent migration in the same epithelial cell background. Adhesion to fibronectin by alpha(v)beta3 supports extensive actin cytoskeletal reorganization through the actin-severing protein cofilin, resulting in a single broad lamellipod with static cell-matrix adhesions at the leading edge. Adhesion by alpha5beta1 instead leads to the phosphorylation/inactivation of cofilin, and these cells fail to polarize their cytoskeleton but extend thin protrusions containing highly dynamic cell-matrix adhesions in multiple directions. The activity of the small GTPase RhoA is particularly high in cells adhering by alpha5beta1, and inhibition of Rho signaling causes a switch from a beta1- to a beta3-associated mode of migration, whereas increased Rho activity has the opposite effect. Thus, alterations in integrin expression profiles allow cells to modulate several critical aspects of the motile machinery through Rho GTPases.

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Expression of activated Rac in GEβ1 cells. (A) GEβ1 cells transiently transfected with RacQ61L in combination with GFP cDNA as a transfection marker (inset) were seeded on FN-coated coverslips 24 h after transfection for 12 h, and were fixed, permeabilized, and stained for F-actin. Arrows indicate transfected cells. Bar, 10 μm. (B) Analysis of GFP-paxillin dynamics in cell–matrix adhesions of GEβ1, GEβ3, and GEβ1 cells transiently transfected with RacQ61L in combination with dsRed cDNA as a transfection marker. Shown is the halftime of loss of fluorescence (τ) ± SEM calculated from FLIP experiments such as depicted in Fig. 4 D. (C) Rac1 and RhoA activity assay in control GEβ1 cells and two stable GEβ1RacQ61L clones. (D) GEβ1, GEβ3, and two GEβ1RacQ61L clones were plated on FN-coated coverslips either sparsely for 2 h followed by treatment with PMA for 1 h (top) or confluently overnight followed by wounding and incubation for an additional 5 h (bottom). Preparations were fixed, permeabilized, and stained for F-actin. Open arrowheads indicate the direction of the wound; filled arrowheads indicate lamellipodia. Bar, 10 μm. Note that GEβ1RacQ61L cells do not show extensive actin cytoskeletal remodeling such as seen in GEβ3 besides increased ruffling in response to PMA or wounding. (E) Control GEβ1 cells and two GEβ1RacQ61L clones were plated on FN-coated coverslips for 1 h. Western blot analysis of total lysates with the indicated antibodies is shown. (F) Mean ± SD of relative cofilin Ser3 phosphorylation determined from two individual experiments such as depicted in E. (G) GEβ1RacQ61L clones were transiently transfected with a plasmid encoding GFP-tagged dominant-active cofilinS3A 24 h before plating on FN-coated dishes. After 2 h of adhesion, cells were stimulated with PMA for 1 h, fixed, permeabilized, and stained for F-actin. Arrows indicate transfected cells. Note ruffling in nontransfected cells versus extensive cytoskeletal reorganization in transfected cells. Bar, 10 μm. Quantification of the percentage of cells responding to PMA treatment by formation of broad lamellipodia is depicted in the graph. Mean ± SD of ∼100 cells analyzed in two independent assays is shown.
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fig6: Expression of activated Rac in GEβ1 cells. (A) GEβ1 cells transiently transfected with RacQ61L in combination with GFP cDNA as a transfection marker (inset) were seeded on FN-coated coverslips 24 h after transfection for 12 h, and were fixed, permeabilized, and stained for F-actin. Arrows indicate transfected cells. Bar, 10 μm. (B) Analysis of GFP-paxillin dynamics in cell–matrix adhesions of GEβ1, GEβ3, and GEβ1 cells transiently transfected with RacQ61L in combination with dsRed cDNA as a transfection marker. Shown is the halftime of loss of fluorescence (τ) ± SEM calculated from FLIP experiments such as depicted in Fig. 4 D. (C) Rac1 and RhoA activity assay in control GEβ1 cells and two stable GEβ1RacQ61L clones. (D) GEβ1, GEβ3, and two GEβ1RacQ61L clones were plated on FN-coated coverslips either sparsely for 2 h followed by treatment with PMA for 1 h (top) or confluently overnight followed by wounding and incubation for an additional 5 h (bottom). Preparations were fixed, permeabilized, and stained for F-actin. Open arrowheads indicate the direction of the wound; filled arrowheads indicate lamellipodia. Bar, 10 μm. Note that GEβ1RacQ61L cells do not show extensive actin cytoskeletal remodeling such as seen in GEβ3 besides increased ruffling in response to PMA or wounding. (E) Control GEβ1 cells and two GEβ1RacQ61L clones were plated on FN-coated coverslips for 1 h. Western blot analysis of total lysates with the indicated antibodies is shown. (F) Mean ± SD of relative cofilin Ser3 phosphorylation determined from two individual experiments such as depicted in E. (G) GEβ1RacQ61L clones were transiently transfected with a plasmid encoding GFP-tagged dominant-active cofilinS3A 24 h before plating on FN-coated dishes. After 2 h of adhesion, cells were stimulated with PMA for 1 h, fixed, permeabilized, and stained for F-actin. Arrows indicate transfected cells. Note ruffling in nontransfected cells versus extensive cytoskeletal reorganization in transfected cells. Bar, 10 μm. Quantification of the percentage of cells responding to PMA treatment by formation of broad lamellipodia is depicted in the graph. Mean ± SD of ∼100 cells analyzed in two independent assays is shown.

Mentions: Extensive cross talk takes place between effector pathways downstream of Rho and Rac. In GEβ1 and GEβ3 cells, the balance between Rho and Rac activities differs due to the higher amounts of Rho-GTP in GEβ1 cells (Fig. 5 A). To test if this balance, rather than RhoA activity by itself, was responsible for the different modes of migration of GEβ1 and GEβ3 cells, we transiently expressed an activated mutant of Rac1 in GEβ1 cells. Expression of RacQ61L caused a conversion from a β1- to a β3-associated morphology (Fig. 6 A). Moreover, the conversion to a GEβ3-like morphology observed in GEβ1 cells transiently expressing RacQ61L was accompanied by a decrease in the dynamics of GFP-paxillin in their cell–matrix adhesions to a level that became indistinguishable from that observed in GEβ3 (Fig. 6 B).


Integrins control motile strategy through a Rho-cofilin pathway.

Danen EH, van Rheenen J, Franken W, Huveneers S, Sonneveld P, Jalink K, Sonnenberg A - J. Cell Biol. (2005)

Expression of activated Rac in GEβ1 cells. (A) GEβ1 cells transiently transfected with RacQ61L in combination with GFP cDNA as a transfection marker (inset) were seeded on FN-coated coverslips 24 h after transfection for 12 h, and were fixed, permeabilized, and stained for F-actin. Arrows indicate transfected cells. Bar, 10 μm. (B) Analysis of GFP-paxillin dynamics in cell–matrix adhesions of GEβ1, GEβ3, and GEβ1 cells transiently transfected with RacQ61L in combination with dsRed cDNA as a transfection marker. Shown is the halftime of loss of fluorescence (τ) ± SEM calculated from FLIP experiments such as depicted in Fig. 4 D. (C) Rac1 and RhoA activity assay in control GEβ1 cells and two stable GEβ1RacQ61L clones. (D) GEβ1, GEβ3, and two GEβ1RacQ61L clones were plated on FN-coated coverslips either sparsely for 2 h followed by treatment with PMA for 1 h (top) or confluently overnight followed by wounding and incubation for an additional 5 h (bottom). Preparations were fixed, permeabilized, and stained for F-actin. Open arrowheads indicate the direction of the wound; filled arrowheads indicate lamellipodia. Bar, 10 μm. Note that GEβ1RacQ61L cells do not show extensive actin cytoskeletal remodeling such as seen in GEβ3 besides increased ruffling in response to PMA or wounding. (E) Control GEβ1 cells and two GEβ1RacQ61L clones were plated on FN-coated coverslips for 1 h. Western blot analysis of total lysates with the indicated antibodies is shown. (F) Mean ± SD of relative cofilin Ser3 phosphorylation determined from two individual experiments such as depicted in E. (G) GEβ1RacQ61L clones were transiently transfected with a plasmid encoding GFP-tagged dominant-active cofilinS3A 24 h before plating on FN-coated dishes. After 2 h of adhesion, cells were stimulated with PMA for 1 h, fixed, permeabilized, and stained for F-actin. Arrows indicate transfected cells. Note ruffling in nontransfected cells versus extensive cytoskeletal reorganization in transfected cells. Bar, 10 μm. Quantification of the percentage of cells responding to PMA treatment by formation of broad lamellipodia is depicted in the graph. Mean ± SD of ∼100 cells analyzed in two independent assays is shown.
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fig6: Expression of activated Rac in GEβ1 cells. (A) GEβ1 cells transiently transfected with RacQ61L in combination with GFP cDNA as a transfection marker (inset) were seeded on FN-coated coverslips 24 h after transfection for 12 h, and were fixed, permeabilized, and stained for F-actin. Arrows indicate transfected cells. Bar, 10 μm. (B) Analysis of GFP-paxillin dynamics in cell–matrix adhesions of GEβ1, GEβ3, and GEβ1 cells transiently transfected with RacQ61L in combination with dsRed cDNA as a transfection marker. Shown is the halftime of loss of fluorescence (τ) ± SEM calculated from FLIP experiments such as depicted in Fig. 4 D. (C) Rac1 and RhoA activity assay in control GEβ1 cells and two stable GEβ1RacQ61L clones. (D) GEβ1, GEβ3, and two GEβ1RacQ61L clones were plated on FN-coated coverslips either sparsely for 2 h followed by treatment with PMA for 1 h (top) or confluently overnight followed by wounding and incubation for an additional 5 h (bottom). Preparations were fixed, permeabilized, and stained for F-actin. Open arrowheads indicate the direction of the wound; filled arrowheads indicate lamellipodia. Bar, 10 μm. Note that GEβ1RacQ61L cells do not show extensive actin cytoskeletal remodeling such as seen in GEβ3 besides increased ruffling in response to PMA or wounding. (E) Control GEβ1 cells and two GEβ1RacQ61L clones were plated on FN-coated coverslips for 1 h. Western blot analysis of total lysates with the indicated antibodies is shown. (F) Mean ± SD of relative cofilin Ser3 phosphorylation determined from two individual experiments such as depicted in E. (G) GEβ1RacQ61L clones were transiently transfected with a plasmid encoding GFP-tagged dominant-active cofilinS3A 24 h before plating on FN-coated dishes. After 2 h of adhesion, cells were stimulated with PMA for 1 h, fixed, permeabilized, and stained for F-actin. Arrows indicate transfected cells. Note ruffling in nontransfected cells versus extensive cytoskeletal reorganization in transfected cells. Bar, 10 μm. Quantification of the percentage of cells responding to PMA treatment by formation of broad lamellipodia is depicted in the graph. Mean ± SD of ∼100 cells analyzed in two independent assays is shown.
Mentions: Extensive cross talk takes place between effector pathways downstream of Rho and Rac. In GEβ1 and GEβ3 cells, the balance between Rho and Rac activities differs due to the higher amounts of Rho-GTP in GEβ1 cells (Fig. 5 A). To test if this balance, rather than RhoA activity by itself, was responsible for the different modes of migration of GEβ1 and GEβ3 cells, we transiently expressed an activated mutant of Rac1 in GEβ1 cells. Expression of RacQ61L caused a conversion from a β1- to a β3-associated morphology (Fig. 6 A). Moreover, the conversion to a GEβ3-like morphology observed in GEβ1 cells transiently expressing RacQ61L was accompanied by a decrease in the dynamics of GFP-paxillin in their cell–matrix adhesions to a level that became indistinguishable from that observed in GEβ3 (Fig. 6 B).

Bottom Line: During wound healing, angiogenesis, and tumor invasion, cells often change their expression profiles of fibronectin-binding integrins.The activity of the small GTPase RhoA is particularly high in cells adhering by alpha5beta1, and inhibition of Rho signaling causes a switch from a beta1- to a beta3-associated mode of migration, whereas increased Rho activity has the opposite effect.Thus, alterations in integrin expression profiles allow cells to modulate several critical aspects of the motile machinery through Rho GTPases.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. e.danen@nki.nl

ABSTRACT
During wound healing, angiogenesis, and tumor invasion, cells often change their expression profiles of fibronectin-binding integrins. Here, we show that beta1 integrins promote random migration, whereas beta3 integrins promote persistent migration in the same epithelial cell background. Adhesion to fibronectin by alpha(v)beta3 supports extensive actin cytoskeletal reorganization through the actin-severing protein cofilin, resulting in a single broad lamellipod with static cell-matrix adhesions at the leading edge. Adhesion by alpha5beta1 instead leads to the phosphorylation/inactivation of cofilin, and these cells fail to polarize their cytoskeleton but extend thin protrusions containing highly dynamic cell-matrix adhesions in multiple directions. The activity of the small GTPase RhoA is particularly high in cells adhering by alpha5beta1, and inhibition of Rho signaling causes a switch from a beta1- to a beta3-associated mode of migration, whereas increased Rho activity has the opposite effect. Thus, alterations in integrin expression profiles allow cells to modulate several critical aspects of the motile machinery through Rho GTPases.

Show MeSH
Related in: MedlinePlus