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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 distribution and dynamics of cell–matrix adhesions. (A) Images of GEβ1 or GEβ3 cells stably expressing GFP-paxillin were taken at the cell–substrate contact area every 15 s after plating on FN-coated coverslips. The time after plating of the first image is indicated (t0). Bars, 10 μm. The right-most panel shows detailed images of the region indicated by arrows at the indicated time points. See supplemental data for the accompanying videos. (B and C) FRAP analysis of GFP-paxillin (B) and GFP-vinculin (C) in cell–matrix adhesions of GEβ1 or GEβ3 cells. Mean ± SEM of three independent experiments, in which at least six cells per experiment were analyzed, is shown. (D) FLIP analysis of GFP-paxillin in cell–matrix adhesions of GEβ1 or GEβ3 cells. Mean ± SEM of four independent experiments, in which 10 adhesions per cell in at least 3 cells per experiment were analyzed, is shown. (E) Western blot using GFP antibody and α-tubulin–loading control antibody on GEβ1 (lanes 1–3) and GEβ3 cells (lanes 4–6) stably expressing GFP-paxillin (lanes 2 and 5), GFP-vinculin (lanes 3 and 6), or controls (lanes 1 and 4). Molecular weights are indicated at the left. See supplemental data for example pictures of FLIP experiments (available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1).
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fig4: β1 and β3 integrins differentially regulate distribution and dynamics of cell–matrix adhesions. (A) Images of GEβ1 or GEβ3 cells stably expressing GFP-paxillin were taken at the cell–substrate contact area every 15 s after plating on FN-coated coverslips. The time after plating of the first image is indicated (t0). Bars, 10 μm. The right-most panel shows detailed images of the region indicated by arrows at the indicated time points. See supplemental data for the accompanying videos. (B and C) FRAP analysis of GFP-paxillin (B) and GFP-vinculin (C) in cell–matrix adhesions of GEβ1 or GEβ3 cells. Mean ± SEM of three independent experiments, in which at least six cells per experiment were analyzed, is shown. (D) FLIP analysis of GFP-paxillin in cell–matrix adhesions of GEβ1 or GEβ3 cells. Mean ± SEM of four independent experiments, in which 10 adhesions per cell in at least 3 cells per experiment were analyzed, is shown. (E) Western blot using GFP antibody and α-tubulin–loading control antibody on GEβ1 (lanes 1–3) and GEβ3 cells (lanes 4–6) stably expressing GFP-paxillin (lanes 2 and 5), GFP-vinculin (lanes 3 and 6), or controls (lanes 1 and 4). Molecular weights are indicated at the left. See supplemental data for example pictures of FLIP experiments (available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1).

Mentions: In addition to actin cytoskeletal polarization, persistent migration also requires cell–matrix adhesions at the leading edge to be sufficiently static to stabilize the lamellipod and generate traction forces. Therefore, we analyzed if differences in the dynamics of cell–matrix adhesions in GEβ1 and GEβ3 could explain the distinct migratory behavior of these cells. Time lapse confocal imaging of GFP-paxillin in migrating GEβ1 cells showed cell–matrix adhesions sliding away from the main cell body in membrane protrusions that extended in multiple directions (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1). These extensions were often short-lived, and their collapse was accompanied by a rapid retraction of the adhesions. By contrast, cell–matrix adhesions at the leading edge of GEβ3 cells were relatively inert, whereas large adhesions were observed to slide inwards at the rear. The same phenomenon could be observed during cell spreading on FN: adhesions in GEβ1 cells were seen sliding outwards, whereas adhesions in GEβ3 did not obviously move. Rather, new adhesions accumulated in GEβ3 cells while existing adhesions remained intact (Fig. 4 A; Video 4). The apparent sliding of adhesions in GEβ1 cells was correlated with a relatively fast turnover rate (within 3–4 min) as compared with that of adhesions in GEβ3 cells (stable for at least 10 min) (Fig. 4 A and unpublished data).


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 distribution and dynamics of cell–matrix adhesions. (A) Images of GEβ1 or GEβ3 cells stably expressing GFP-paxillin were taken at the cell–substrate contact area every 15 s after plating on FN-coated coverslips. The time after plating of the first image is indicated (t0). Bars, 10 μm. The right-most panel shows detailed images of the region indicated by arrows at the indicated time points. See supplemental data for the accompanying videos. (B and C) FRAP analysis of GFP-paxillin (B) and GFP-vinculin (C) in cell–matrix adhesions of GEβ1 or GEβ3 cells. Mean ± SEM of three independent experiments, in which at least six cells per experiment were analyzed, is shown. (D) FLIP analysis of GFP-paxillin in cell–matrix adhesions of GEβ1 or GEβ3 cells. Mean ± SEM of four independent experiments, in which 10 adhesions per cell in at least 3 cells per experiment were analyzed, is shown. (E) Western blot using GFP antibody and α-tubulin–loading control antibody on GEβ1 (lanes 1–3) and GEβ3 cells (lanes 4–6) stably expressing GFP-paxillin (lanes 2 and 5), GFP-vinculin (lanes 3 and 6), or controls (lanes 1 and 4). Molecular weights are indicated at the left. See supplemental data for example pictures of FLIP experiments (available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1).
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Related In: Results  -  Collection

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fig4: β1 and β3 integrins differentially regulate distribution and dynamics of cell–matrix adhesions. (A) Images of GEβ1 or GEβ3 cells stably expressing GFP-paxillin were taken at the cell–substrate contact area every 15 s after plating on FN-coated coverslips. The time after plating of the first image is indicated (t0). Bars, 10 μm. The right-most panel shows detailed images of the region indicated by arrows at the indicated time points. See supplemental data for the accompanying videos. (B and C) FRAP analysis of GFP-paxillin (B) and GFP-vinculin (C) in cell–matrix adhesions of GEβ1 or GEβ3 cells. Mean ± SEM of three independent experiments, in which at least six cells per experiment were analyzed, is shown. (D) FLIP analysis of GFP-paxillin in cell–matrix adhesions of GEβ1 or GEβ3 cells. Mean ± SEM of four independent experiments, in which 10 adhesions per cell in at least 3 cells per experiment were analyzed, is shown. (E) Western blot using GFP antibody and α-tubulin–loading control antibody on GEβ1 (lanes 1–3) and GEβ3 cells (lanes 4–6) stably expressing GFP-paxillin (lanes 2 and 5), GFP-vinculin (lanes 3 and 6), or controls (lanes 1 and 4). Molecular weights are indicated at the left. See supplemental data for example pictures of FLIP experiments (available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1).
Mentions: In addition to actin cytoskeletal polarization, persistent migration also requires cell–matrix adhesions at the leading edge to be sufficiently static to stabilize the lamellipod and generate traction forces. Therefore, we analyzed if differences in the dynamics of cell–matrix adhesions in GEβ1 and GEβ3 could explain the distinct migratory behavior of these cells. Time lapse confocal imaging of GFP-paxillin in migrating GEβ1 cells showed cell–matrix adhesions sliding away from the main cell body in membrane protrusions that extended in multiple directions (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1). These extensions were often short-lived, and their collapse was accompanied by a rapid retraction of the adhesions. By contrast, cell–matrix adhesions at the leading edge of GEβ3 cells were relatively inert, whereas large adhesions were observed to slide inwards at the rear. The same phenomenon could be observed during cell spreading on FN: adhesions in GEβ1 cells were seen sliding outwards, whereas adhesions in GEβ3 did not obviously move. Rather, new adhesions accumulated in GEβ3 cells while existing adhesions remained intact (Fig. 4 A; Video 4). The apparent sliding of adhesions in GEβ1 cells was correlated with a relatively fast turnover rate (within 3–4 min) as compared with that of adhesions in GEβ3 cells (stable for at least 10 min) (Fig. 4 A and unpublished data).

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