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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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Inhibition of Rho signaling in GEβ1 cells. (A) Rac and Rho activity assay in GEβ1 and GEβ3 cells. (B) GEβ1 or GEβ3 cells transiently transfected with the indicated expression plasmids in combination with GFP cDNA as a transfection marker (insets) 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. Bars, 10 μm. (C) Analysis of GFP-paxillin dynamics in cell–matrix adhesions of GEβ1, GEβ3, and Y27632-treated GEβ1 cells. Shown is the halftime of loss of fluorescence (τ) ± SEM calculated from FLIP experiments such as depicted in Fig. 4 D. (D) Control or C3-transfected GEβ1 cells (indicated by GFP; inset and arrow) were plated overnight on FN-coated coverslips and stimulated with PMA for 1 h in the absence or presence of Y27632 as indicated. Preparations were fixed, permeabilized, and stained for F-actin. Filled arrowheads indicate Y27632-induced membrane ruffles/lamellipodia. Bars, 5 μm. (E) GEβ1 cells were plated overnight on FN-coated coverslips, confluent monolayers were wounded with a micropipette tip, and preparations were fixed, permeabilized, and stained for F-actin after 5 h incubation in the absence or presence of Y27632. Open arrowheads indicate the direction of the wound; filled arrowheads indicate Y27632-induced protrusions of cells moving into the wounded area. Bar, 10 μm. (F) GEβ1 cells were plated on FN-coated coverslips for the indicated times in the absence or presence of Y27632 as indicated. Western blot analysis of total lysates with the indicated antibodies is shown. (G) Mean ± SD of relative cofilin Ser3 phosphorylation determined from two individual experiments such as depicted in F.
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fig5: Inhibition of Rho signaling in GEβ1 cells. (A) Rac and Rho activity assay in GEβ1 and GEβ3 cells. (B) GEβ1 or GEβ3 cells transiently transfected with the indicated expression plasmids in combination with GFP cDNA as a transfection marker (insets) 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. Bars, 10 μm. (C) Analysis of GFP-paxillin dynamics in cell–matrix adhesions of GEβ1, GEβ3, and Y27632-treated GEβ1 cells. Shown is the halftime of loss of fluorescence (τ) ± SEM calculated from FLIP experiments such as depicted in Fig. 4 D. (D) Control or C3-transfected GEβ1 cells (indicated by GFP; inset and arrow) were plated overnight on FN-coated coverslips and stimulated with PMA for 1 h in the absence or presence of Y27632 as indicated. Preparations were fixed, permeabilized, and stained for F-actin. Filled arrowheads indicate Y27632-induced membrane ruffles/lamellipodia. Bars, 5 μm. (E) GEβ1 cells were plated overnight on FN-coated coverslips, confluent monolayers were wounded with a micropipette tip, and preparations were fixed, permeabilized, and stained for F-actin after 5 h incubation in the absence or presence of Y27632. Open arrowheads indicate the direction of the wound; filled arrowheads indicate Y27632-induced protrusions of cells moving into the wounded area. Bar, 10 μm. (F) GEβ1 cells were plated on FN-coated coverslips for the indicated times in the absence or presence of Y27632 as indicated. Western blot analysis of total lysates with the indicated antibodies is shown. (G) Mean ± SD of relative cofilin Ser3 phosphorylation determined from two individual experiments such as depicted in F.

Mentions: These findings indicate that the type of integrin involved in the adhesion to FN strongly affects cell–matrix adhesion dynamics. To further test this, we analyzed the dynamics of two different cell–matrix adhesion components—paxillin and vinculin—in more detail by performing fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments. Strikingly, even in stationary, fully spread cells, the dynamics of GFP-paxillin and GFP-vinculin in adhesions of GEβ1 cells as measured by FRAP were significantly higher than those in adhesions formed by GEβ3 cells (P = 0.0003 and P < 0.0001, respectively; Fig. 4, B and C). These findings were confirmed using FLIP as an alternative method of analysis: loss of fluorescence from cell–matrix adhesions in GEβ1 cells expressing GFP-paxillin was significantly faster (P < 0.0001) than that in GEβ3 cells (time required for a 50% reduction in fluorescence, τ = 5.6 min in GEβ1 vs. τ = 21.3 min in GEβ3) (Fig. 4 D, Fig. 5 C; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1). The results were not affected by differences in expression levels or degradation of the GFP fusion constructs as shown by Western blot analysis (Fig. 4 E).


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)

Inhibition of Rho signaling in GEβ1 cells. (A) Rac and Rho activity assay in GEβ1 and GEβ3 cells. (B) GEβ1 or GEβ3 cells transiently transfected with the indicated expression plasmids in combination with GFP cDNA as a transfection marker (insets) 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. Bars, 10 μm. (C) Analysis of GFP-paxillin dynamics in cell–matrix adhesions of GEβ1, GEβ3, and Y27632-treated GEβ1 cells. Shown is the halftime of loss of fluorescence (τ) ± SEM calculated from FLIP experiments such as depicted in Fig. 4 D. (D) Control or C3-transfected GEβ1 cells (indicated by GFP; inset and arrow) were plated overnight on FN-coated coverslips and stimulated with PMA for 1 h in the absence or presence of Y27632 as indicated. Preparations were fixed, permeabilized, and stained for F-actin. Filled arrowheads indicate Y27632-induced membrane ruffles/lamellipodia. Bars, 5 μm. (E) GEβ1 cells were plated overnight on FN-coated coverslips, confluent monolayers were wounded with a micropipette tip, and preparations were fixed, permeabilized, and stained for F-actin after 5 h incubation in the absence or presence of Y27632. Open arrowheads indicate the direction of the wound; filled arrowheads indicate Y27632-induced protrusions of cells moving into the wounded area. Bar, 10 μm. (F) GEβ1 cells were plated on FN-coated coverslips for the indicated times in the absence or presence of Y27632 as indicated. Western blot analysis of total lysates with the indicated antibodies is shown. (G) Mean ± SD of relative cofilin Ser3 phosphorylation determined from two individual experiments such as depicted in F.
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getmorefigures.php?uid=PMC2171933&req=5

fig5: Inhibition of Rho signaling in GEβ1 cells. (A) Rac and Rho activity assay in GEβ1 and GEβ3 cells. (B) GEβ1 or GEβ3 cells transiently transfected with the indicated expression plasmids in combination with GFP cDNA as a transfection marker (insets) 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. Bars, 10 μm. (C) Analysis of GFP-paxillin dynamics in cell–matrix adhesions of GEβ1, GEβ3, and Y27632-treated GEβ1 cells. Shown is the halftime of loss of fluorescence (τ) ± SEM calculated from FLIP experiments such as depicted in Fig. 4 D. (D) Control or C3-transfected GEβ1 cells (indicated by GFP; inset and arrow) were plated overnight on FN-coated coverslips and stimulated with PMA for 1 h in the absence or presence of Y27632 as indicated. Preparations were fixed, permeabilized, and stained for F-actin. Filled arrowheads indicate Y27632-induced membrane ruffles/lamellipodia. Bars, 5 μm. (E) GEβ1 cells were plated overnight on FN-coated coverslips, confluent monolayers were wounded with a micropipette tip, and preparations were fixed, permeabilized, and stained for F-actin after 5 h incubation in the absence or presence of Y27632. Open arrowheads indicate the direction of the wound; filled arrowheads indicate Y27632-induced protrusions of cells moving into the wounded area. Bar, 10 μm. (F) GEβ1 cells were plated on FN-coated coverslips for the indicated times in the absence or presence of Y27632 as indicated. Western blot analysis of total lysates with the indicated antibodies is shown. (G) Mean ± SD of relative cofilin Ser3 phosphorylation determined from two individual experiments such as depicted in F.
Mentions: These findings indicate that the type of integrin involved in the adhesion to FN strongly affects cell–matrix adhesion dynamics. To further test this, we analyzed the dynamics of two different cell–matrix adhesion components—paxillin and vinculin—in more detail by performing fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments. Strikingly, even in stationary, fully spread cells, the dynamics of GFP-paxillin and GFP-vinculin in adhesions of GEβ1 cells as measured by FRAP were significantly higher than those in adhesions formed by GEβ3 cells (P = 0.0003 and P < 0.0001, respectively; Fig. 4, B and C). These findings were confirmed using FLIP as an alternative method of analysis: loss of fluorescence from cell–matrix adhesions in GEβ1 cells expressing GFP-paxillin was significantly faster (P < 0.0001) than that in GEβ3 cells (time required for a 50% reduction in fluorescence, τ = 5.6 min in GEβ1 vs. τ = 21.3 min in GEβ3) (Fig. 4 D, Fig. 5 C; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1). The results were not affected by differences in expression levels or degradation of the GFP fusion constructs as shown by Western blot analysis (Fig. 4 E).

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