Integrins control motile strategy through a Rho-cofilin pathway

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.

Figure 1.

β1 and β3 integrins differentially affect motile behavior. (A and B) Migration tracks of GEβ1 (A) or GEβ3 cells (B) seeded sparsely on FN-coated coverslips and followed for 16 h. Shown are 20 cells obtained from three independent experiments. (C) Analysis of persistence (ratio of the direct distance from start point to end point divided by the total track distance) and speed of migrating GEβ1 and GEβ3 cells in sparse cultures. (D) Analysis of MTOC polarization in wounding assays with GEβ1 (filled bars) and GEβ3 cells (open bars) at the indicated time points after wounding of confluent monolayers on FN-coated coverslips. Mean ± SD of ∼100 cells analyzed in three independent assays is shown. See supplemental data for the accompanying videos and immunofluorescence images (available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1).

Figure 2.

β1 and β3 integrins differentially regulate actin cytoskeletal reorganization. (A) GEβ1 or GEβ3 cells, stably expressing GFP-paxillin, were plated overnight on FN-coated coverslips, confluent monolayers were wounded with a micropipette tip, and preparations were fixed and permeabilized after 5 h. Organization of the actin cytoskeleton and localization of paxillin is shown as indicated. Arrowheads indicate protrusions of cells moving into the wounded area. Bar, 10 μm. (B) GEβ1 and GEβ3 cells were plated on FN-coated coverslips for 4 h and fixed and permeabilized either immediately (control) or after stimulation with PMA for 1 h as indicated. Single staining for F-actin, double staining for F-actin (red) and paxillin (green), or single staining for α-tubulin are shown as indicated with details of membrane protrusions shown at higher magnification at the far right. Dotted line separates two different protrusions. Bars, 5 μm.

Figure 3.

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.

Figure 4.

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).

Figure 5.

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.

Figure 6.

Expression of activated Rac in GEβ1 cells. (A) GEβ1 cells transiently transfected with Rac^Q61L 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 Rac^Q61L 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 GEβ1Rac^Q61L clones. (D) GEβ1, GEβ3, and two GEβ1Rac^Q61L 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 GEβ1Rac^Q61L 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 GEβ1Rac^Q61L 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) GEβ1Rac^Q61L clones were transiently transfected with a plasmid encoding GFP-tagged dominant-active cofilin^S3A 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.

Figure 7.

Model for the control of cell migration by integrin-specific regulation of Rho GTPases. Adhesion by β1 integrins promotes strong Rho/Rho kinase signaling. This counteracts three important parameters of persistent cell migration: (1) Rac-mediated lamellipodia formation; (2) development of static cell–matrix adhesions; and (3) cofilin-mediated actin cytoskeletal reorganization. As a result, β1 integrins promote a random mode of migration. Inhibition of Rho/Rho kinase signaling relieves the suppression of all three aspects and causes a switch from β1- to β3-associated behavior. Conversely, increased Rho signaling in cells adhering by αvβ3 triggers a conversion to β1-associated behavior. Increased Rac signaling can also stimulate a partial conversion from β1- to β3-associated behavior with increased lamellipodia formation and stabilization of cell–matrix adhesions. However, this does not lead to increased cofilin activity and hence, does not stimulate persistence.