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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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β1 and β3 integrins differentially regulate cofilin pSer3 levels. (A) GEβ1 and GEβ3 cells were serum starved overnight, trypsinized, incubated in suspension for 30–60 min, and plated on FN for the indicated times (left and middle) or followed by plating on FN for 90 min and subsequent treatment with PMA for the indicated times (right). Western blot analysis of total lysates with the indicated antibodies is shown. (B) Quantification based on four (left) or two (right) experiments such as shown in A. Mean ± SD of relative cofilin Ser3 phosphorylation in GEβ1 (filled bars) and GEβ3 cells (open bars) is shown. (C) GEβ3 cells transiently transfected with a cDNA encoding GFP-cofilinS3E were plated on FN-coated coverslips for 4 h and fixed and permeabilized after stimulation with PMA for 1 h. Organization of the actin cytoskeleton is shown. Inset shows GFP signal. Note that the upper, nontransfected cell generates a typical broad lamellipod whereas transfected cells do not. Bar, 10 μm. Quantification of the percentage of cells responding to PMA treatment by formation of broad lamellipodia is depicted in the graph at the right. Mean ± SD of ∼100 cells analyzed in two independent assays is shown.
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fig3: β1 and β3 integrins differentially regulate cofilin pSer3 levels. (A) GEβ1 and GEβ3 cells were serum starved overnight, trypsinized, incubated in suspension for 30–60 min, and plated on FN for the indicated times (left and middle) or followed by plating on FN for 90 min and subsequent treatment with PMA for the indicated times (right). Western blot analysis of total lysates with the indicated antibodies is shown. (B) Quantification based on four (left) or two (right) experiments such as shown in A. Mean ± SD of relative cofilin Ser3 phosphorylation in GEβ1 (filled bars) and GEβ3 cells (open bars) is shown. (C) GEβ3 cells transiently transfected with a cDNA encoding GFP-cofilinS3E were plated on FN-coated coverslips for 4 h and fixed and permeabilized after stimulation with PMA for 1 h. Organization of the actin cytoskeleton is shown. Inset shows GFP signal. Note that the upper, nontransfected cell generates a typical broad lamellipod whereas transfected cells do not. Bar, 10 μm. Quantification of the percentage of cells responding to PMA treatment by formation of broad lamellipodia is depicted in the graph at the right. Mean ± SD of ∼100 cells analyzed in two independent assays is shown.

Mentions: Cofilin has been implicated in cytoskeletal polarization and directional migration (Dawe et al., 2003; Ghosh et al., 2004; Mouneimne et al., 2004). We tested whether the observed differences in actin cytoskeletal reorganization in wounded or PMA-treated GEβ1 and GEβ3 cultures were related to differences in cofilin activity. Phospho-cofilin levels in serum-starved, suspended cells varied between experiments (Fig. 3 A; compare time “0” in left and middle). Nevertheless, irrespective of the phosphorylation status before plating, the relative amount of cofilin phosphorylated on Ser3 in cells attached to FN was much higher in GEβ1 than in GEβ3 cells, indicating that FN-adhered GEβ3 cells contained a larger pool of nonphosphorylated, active cofilin (Fig. 3 A, left and middle; Fig. 3 B, left). Notably, in line with the observation that PMA hardly affected the morphology of GEβ1 cells, PMA treatment did not affect the cofilin phosphorylation status (i.e., no decrease in cofilin Ser3 phosphorylation was observed in GEβ1 cells treated with PMA; Fig. 3, A and B, right). Finally, to analyze if the difference in cofilin activity could explain the differences in actin reorganization, we expressed dominant-active and -inactive cofilin mutants in GEβ1 and GEβ3 cells, respectively. Expression of a GFP-tagged active, nonphosphorylatable cofilinS3A mutant in GEβ1 cells or expression of an inactive, phospho-mimetic cofilinS3E mutant in GEβ3 cells had no apparent effect on cell–matrix adhesion distribution or actin cytoskeletal organization in cells plated for 2–4 h on FN (unpublished data). GEβ1 cells transfected with cofilinS3A also remained unable to reorganize their actin cytoskeleton in response to PMA (unpublished data). However, expression of the cofilinS3E mutant in GEβ3 significantly interfered with the formation of broad lamellipodia in response to PMA, indicating that cofilin activity is required for the actin cytoskeletal reorganization (Fig. 3 C).


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)

β1 and β3 integrins differentially regulate cofilin pSer3 levels. (A) GEβ1 and GEβ3 cells were serum starved overnight, trypsinized, incubated in suspension for 30–60 min, and plated on FN for the indicated times (left and middle) or followed by plating on FN for 90 min and subsequent treatment with PMA for the indicated times (right). Western blot analysis of total lysates with the indicated antibodies is shown. (B) Quantification based on four (left) or two (right) experiments such as shown in A. Mean ± SD of relative cofilin Ser3 phosphorylation in GEβ1 (filled bars) and GEβ3 cells (open bars) is shown. (C) GEβ3 cells transiently transfected with a cDNA encoding GFP-cofilinS3E were plated on FN-coated coverslips for 4 h and fixed and permeabilized after stimulation with PMA for 1 h. Organization of the actin cytoskeleton is shown. Inset shows GFP signal. Note that the upper, nontransfected cell generates a typical broad lamellipod whereas transfected cells do not. Bar, 10 μm. Quantification of the percentage of cells responding to PMA treatment by formation of broad lamellipodia is depicted in the graph at the right. Mean ± SD of ∼100 cells analyzed in two independent assays is shown.
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fig3: β1 and β3 integrins differentially regulate cofilin pSer3 levels. (A) GEβ1 and GEβ3 cells were serum starved overnight, trypsinized, incubated in suspension for 30–60 min, and plated on FN for the indicated times (left and middle) or followed by plating on FN for 90 min and subsequent treatment with PMA for the indicated times (right). Western blot analysis of total lysates with the indicated antibodies is shown. (B) Quantification based on four (left) or two (right) experiments such as shown in A. Mean ± SD of relative cofilin Ser3 phosphorylation in GEβ1 (filled bars) and GEβ3 cells (open bars) is shown. (C) GEβ3 cells transiently transfected with a cDNA encoding GFP-cofilinS3E were plated on FN-coated coverslips for 4 h and fixed and permeabilized after stimulation with PMA for 1 h. Organization of the actin cytoskeleton is shown. Inset shows GFP signal. Note that the upper, nontransfected cell generates a typical broad lamellipod whereas transfected cells do not. Bar, 10 μm. Quantification of the percentage of cells responding to PMA treatment by formation of broad lamellipodia is depicted in the graph at the right. Mean ± SD of ∼100 cells analyzed in two independent assays is shown.
Mentions: Cofilin has been implicated in cytoskeletal polarization and directional migration (Dawe et al., 2003; Ghosh et al., 2004; Mouneimne et al., 2004). We tested whether the observed differences in actin cytoskeletal reorganization in wounded or PMA-treated GEβ1 and GEβ3 cultures were related to differences in cofilin activity. Phospho-cofilin levels in serum-starved, suspended cells varied between experiments (Fig. 3 A; compare time “0” in left and middle). Nevertheless, irrespective of the phosphorylation status before plating, the relative amount of cofilin phosphorylated on Ser3 in cells attached to FN was much higher in GEβ1 than in GEβ3 cells, indicating that FN-adhered GEβ3 cells contained a larger pool of nonphosphorylated, active cofilin (Fig. 3 A, left and middle; Fig. 3 B, left). Notably, in line with the observation that PMA hardly affected the morphology of GEβ1 cells, PMA treatment did not affect the cofilin phosphorylation status (i.e., no decrease in cofilin Ser3 phosphorylation was observed in GEβ1 cells treated with PMA; Fig. 3, A and B, right). Finally, to analyze if the difference in cofilin activity could explain the differences in actin reorganization, we expressed dominant-active and -inactive cofilin mutants in GEβ1 and GEβ3 cells, respectively. Expression of a GFP-tagged active, nonphosphorylatable cofilinS3A mutant in GEβ1 cells or expression of an inactive, phospho-mimetic cofilinS3E mutant in GEβ3 cells had no apparent effect on cell–matrix adhesion distribution or actin cytoskeletal organization in cells plated for 2–4 h on FN (unpublished data). GEβ1 cells transfected with cofilinS3A also remained unable to reorganize their actin cytoskeleton in response to PMA (unpublished data). However, expression of the cofilinS3E mutant in GEβ3 significantly interfered with the formation of broad lamellipodia in response to PMA, indicating that cofilin activity is required for the actin cytoskeletal reorganization (Fig. 3 C).

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