Rab-coupling protein coordinates recycling of alpha5beta1 integrin and EGFR1 to promote cell migration in 3D microenvironments

Abstract

Here we show that blocking the adhesive function of alphavbeta3 integrin with soluble RGD ligands, such as osteopontin or cilengitide, promoted association of Rab-coupling protein (RCP) with alpha5beta1 integrin and drove RCP-dependent recycling of alpha5beta1 to the plasma membrane and its mobilization to dynamic ruffling protrusions at the cell front. These RCP-driven changes in alpha5beta1 trafficking led to acquisition of rapid/random movement on two-dimensional substrates and to a marked increase in fibronectin-dependent migration of tumor cells into three-dimensional matrices. Recycling of alpha5beta1 integrin did not affect its regulation or ability to form adhesive bonds with substrate fibronectin. Instead, alpha5beta1 controlled the association of EGFR1 with RCP to promote the coordinate recycling of these two receptors. This modified signaling downstream of EGFR1 to increase its autophosphorylation and activation of the proinvasive kinase PKB/Akt. We conclude that RCP provides a scaffold that promotes the physical association and coordinate trafficking of alpha5beta1 and EGFR1 and that this drives migration of tumor cells into three-dimensional matrices.

Figure 1.

Inhibition of αvβ3 promotes α5β1 recycling and association of RCP with α5β1. (A) A2780 cells were transfected with a nontargeting shRNA (sh-CON) or a short hairpin–targeting β3 integrin (sh-β3; right) or were left untransfected (left). Cells were surface labeled with 0.2 mg/ml NHS-S-S-Biotin at 4°C and internalization was then allowed to proceed for 30 min at 37°C. Biotin remaining at the cell surface was removed by exposure to MesNa at 4°C, and internalized integrin was chased back to the cell surface at 37°C for the indicated times in the absence (BAS) or presence (CIL) of 1 μM cilengitide, 1 μM c-RADfV, 4.4 μM cRGDfV, or 0.5 μg/ml soluble osteopontin. Cells were then reexposed to MesNa and biotinylated integrin was determined by capture ELISA using microtitre wells coated with anti-human α5 integrin monoclonal antibodies. The proportion of integrin recycled to the plasma membrane is expressed as a percentage of the pool of integrin labeled during the internalization period. (left) Values are mean ± SEM; n = 7 (BAS), n = 4 (CIL), and n = 3 (cRADfV, cRGDfV, and OSP) independent experiments. (right) Values are mean ± SEM; n = 3 independent experiments. (B) A2780 cells were transfected with plasmids encoding GFP or GFP fused to the class I FIPs (GFP-RCP, GFP-FIP2, and GFP-Rip11). Transfected cells were incubated in the absence (BAS) or presence (CIL) of 1 μM cilengitide for 20 min and lysed in a buffer containing 0.15% Tween-20. GFP and GFP fusion proteins were immunoprecipitated from lysates by incubation with beads coupled to a monoclonal antibody recognizing GFP, and these immunoprecipitates analyzed for the presence of α5β1 and αvβ3 by immunoblotting with antibodies recognizing the integrin α5 and β3 chains, respectively. The loading of GFP and GFP fusions was confirmed by immunoblotting with an antibody recognizing GFP (bottom). (C) Cells were transfected with GFP-RCP (top) or left untransfected (bottom), and then incubated in the absence (BAS) or presence (CIL) of 1 μM cilengitide for 20 min. Lysates were immunoprecipitated using monoclonal antibodies recognizing α5β1 integrin (anti-α5), αvβ3 integrin (anti-β3), or an isotype-matched control antibody (RG-16). The presence of α5 and β3 integrin, GFP-RCP, and endogenous RCP were detected by immunoblotting. (D) After cotransfection of GFP or GFP-RCP with either nontargeting shRNA (shRNA-CON) or a short hairpin targeting β3 integrin (shRNA-β3), A2780 cells were incubated in the absence (BAS) or presence (CIL) of 1 μM cilengitide or 0.5 μg/ml osteopontin (OSP) for 20 min. Cells were lysed and anti-GFP immunoprecipitates probed for the presence of α5β1 and GFP as in B. (E) A2780 cells were transfected with GFP, GFP-RCP^wt, or GFP-RCP^621E, treated with cilengitide, and then lysed. Anti-GFP immunoprecipitates were probed for α5β1 and GFP as in B.

Figure 2.

Cilengitide and osteopontin drive alterations in α5β1 recycling and cell migration that are dependent on RCP. (A) A2780 cells were transfected with a nontargeting shRNA (shRNA-CON), a short hairpin targeting RCP (shRNA-RCP), GFP-RCP^wt, or dominant-negative GFP-RCP^621E. Recycling of α5β1 integrin was determined in the presence and absence of 1 μM cilengitide as for Fig. 1 A. Values are mean ± SEM; n = 3 independent experiments. (B) Cells were transfected with GFP-RCP^wt or GFP-RCP^621E and allowed to grow to confluence. Confluent monolayers were wounded with a plastic pipette tip and the cells were allowed to migrate into the wound in the absence (Basal) or presence of 1 μM cilengitide (Cil) or 0.5 μg/ml osteopontin (Osp). The cells were observed by time-lapse video microscopy, the movement of individual cells followed using cell tracking software, and this is presented as overlays of representative trajectories described by cells during the first 9 h of their migration into the wound. The starting position of each cell is denoted by the red dot. Examples of time-lapse movies that correspond to these experiments are included as Videos 1–3 as indicated (available at http://www.jcb.org/cgi/content/full/jcb.200804140/DC1). The persistence and speed of migration were extracted from the track plots. Persistence is defined as the ratio of the vectorial distance traveled to the total path length described by the cell. Values are mean ± SEM; n = 3 independent experiments. Bar, 50 μm.

Figure 3.

Inhibition of αvβ3 promotes the formation of dynamic actin-rich protrusions at the cell front. (A) Confluent monolayers were wounded with a plastic pipette tip and the were cells allowed to migrate for 2 h into the wound in the absence (BASAL) or presence of 1 μM cilengitide (CIL) or 0.5 μg/ml osteopontin (OSP). Cells were fixed and permeabilized and F-actin was visualized using fluorescently conjugated phalloidin. 3D reconstructions were generated from serial Z sections captured using a confocal microscope. Yellow arrow denotes the direction of cell migration. Bar, 10 μm. Time-lapse movies indicating the dynamics of GFP-actin under conditions corresponding to the experiments presented in A are included as Videos 4–6, respectively (available at http://www.jcb.org/cgi/content/full/jcb.200804140/DC1). (B) A2780 cells were transfected with GFP-RCP^wt, GFP-RCP^621E, GFP-FIP2^480E, or GFP-Rip11^630E. Confluent monolayers were wounded with a plastic pipette tip and the cells were allowed to migrate for 2 h into the wound in the absence or presence of 1 μM cilengitide. Cells were fixed and permeabilized and GFP and F-actin was visualized by fluorescence microscopy. 3D reconstructions were generated from serial Z sections captured using a confocal microscope. Yellow arrow denotes the direction of cell migration. Bar, 10 μm.

Figure 4.

Inhibition of αvβ3 promotes migration into matrigel that is dependent on RCP and engagement of α5β1 with FN. (A) Migration of A2780 cells expressing a nontargeting shRNA (shRNA-Con) or a short hairpin targeting β3 integrin (shRNA-β3) into a matrigel plug in the presence and absence of 25 μg/ml FN was determined using an inverted matrigel plug assay. 1 μM cilengitide (CIL) and 0.5 μg/ml osteopontin (OSP) were added as indicated to the matrigel and to both the upper and lower chambers of the inverted matrigel plug assay. Invading cells were stained with Calcein AM and visualized by confocal microscopy. Serial optical sections were captured at 15-μm intervals and are presented as a sequence in which the individual optical sections are placed alongside one another with increasing depth from left to right as indicated. Migration was quantitated by measuring the fluorescence intensity of cells penetrating the matrigel to depths of 45 μm and greater and expressing this as a percentage of the total fluorescence intensity of all cells within the plug. Data represents mean ± SEM from three independent experiments. (B) The involvement of α5β1 integrin in migration driven by addition of cilengitide (left and right) or induced by suppression of αvβ3 levels (middle) was determined by addition of the indicated integrin- and FN-blocking antibodies (left and middle) and also by RNAi of the α5 and β1 subunits of α5β1 (right). Migration was quantified as for A. Values are mean ± SEM from three independent experiments. (C) The migration of A2780 cells expressing nontargeting shRNA (shRNA-CON), a short hairpin targeting RCP integrin (shRNA-RCP), GFP, GFP-RCP^wt, or GFP-RCP^621E into FN-containing matrigel was determined and quantified as for A.

Figure 5.

Cilengitide drives RCP to the tips of extending pseudopods. (A and B) Cells were transfected with GFP-RCP^wt or GFP-RCP^621E and plated onto cell-derived matrix in the presence and absence of 1 μM cilengitide ∼4 h before time-lapse microscopy. Images were captured every 5 min over a 6-h period and movies were generated from these (Videos 7–9) and stills from these movies are presented. Bar, 100 μm. (B) Speed and persistence of migration were determined as for Fig. 3 B, values are mean ± SEM; n = 3 independent experiments. To obtain a measure of pseudopod length, the distance between the center of the nucleus and the cell front (with respect to the direction of migration) was measured using ImageJ. Data represents mean ± SEM; n = 3 independent experiments. (C) Cells were transfected with GFP-RCP, GFP-RCP^621E, or GFP-RCP^379-649 and plated onto cell-derived matrix in the presence and absence of 1 μM cilengitide and imaged by confocal microscopy. Images were captured at 1 frame/second over a period of 100 s and movies were generated from these. Single section confocal image stills corresponding to individual frames from these movies are presented. Bar, 20 μm. The yellow arrows indicate the direction of migration and the portion of the cell within the white square is presented as Video 10. Videos are available at http://www.jcb.org/cgi/content/full/jcb.200804140/DC1.

Figure 6.

RCP does not affect integrin ligand-binding ability but mediates an association between α5β1 and EGFR1. (A and B) A2780 cells were transfected with either RCP^wt or the dominant-negative RCP^621E and incubated for 24 h ±1 μM cilengitide for the last 16 h. Adhesion to FN was analyzed using the spinning disc. (A) The analysis of cell density at 61 points for each treatment as a function of wall shear stress (τ₅₀) is shown. The associated curve fit (solid lines) R² values for the fits are 0.95 (RCP^wt with cilengitide) and 0.92 (RCP^621E with cilengitide). The curve fits were used to extract a mean shear stress for cell detachment (τ₅₀) that is proportional to the number of adhesive integrin–ligand bonds (Boettiger, 2007). (B) The combined analyses for τ₅₀ from three independent experiments run in triplicate are shown; normalized percentages are on the bars. Values are mean ± SEM (n = 8 or 9). (C) Cells were transfected with hairpin vectors targeting β3 integrin, α5 integrin, or EGFR1 in combination with GFP, GFP-RCP^wt, or GFP-RCP^621E (left), or were left untransfected (right). After incubation with 1 μM cilengitide (CIL) or 0.5 μg/ml osteopontin (OSP), cells were lysed and GFP-RCP or α5β1 was immunoprecipitated from the lysates using monoclonal antibodies recognizing GFP (anti-GFP), α5 integrin (anti-α5), or an isotype-matched control antibody (RG-16). The presence of α5 integrin, GFP-RCP, endogenous RCP, and EGFR1 in the immunoprecipitates was then detected by immunoblotting. (D–F) A2780 cells were transfected with a short hairpin–targeting β3 integrin (shRNA-β3; F) or were left untransfected (D and E). Confluent monolayers were wounded and the cells were allowed to migrate into the wound in the absence (D and F) or presence (E) of 1 μM cilengitide. The distribution of EGFR1 (green), RCP (red), and F-actin (blue) was visualized by immunofluorescence followed by confocal microscopy. Bar, 30 μm.

Figure 7.

α5β1 is required for recycling of EGFR1; and uncoupling EGFR1 recycling from α5β1 inhibits migration in 3D. (A–C) Cells were transfected with short hairpin vectors targeting β3, α5, β1, RCP, or EGFR1 or with truncated GFP-RCP^379-649 as indicated. The receptor recycling protocol was then followed as for Fig. 1 A but with biotinylated EGFR1 or α5β1 integrin being determined by capture ELISA using microtitre wells coated with anti-EGFR1 or α5 integrin monoclonal antibodies as indicated by the y-axis labeling of the graphs. The proportion of EGFR1 or integrin recycled to the plasma membrane is expressed as a percentage of the pool of integrin labeled during the internalization period. Values are mean ± SEM; n = 3 or 4 independent experiments. (D) Migration of A2780 cells expressing GFP, GFP-RCP^wt, or GFP-RCP^379-649 into matrigel plugs containing 25 μg/ml FN. 1 μM cilengitide (CIL) was added as indicated to the matrigel and to both the upper and lower chambers of the inverted invasion assay. Migrating cells were detected and quantified as for Fig. 4. Values are mean ± SEM for three independent experiments.

Figure 8.

Osteopontin enhances EGFR1 signaling via a mechanism that requires α5β1, RCP, and association of EGFR1 with RCP. Cells were transfected with short hairpin vectors targeting β3, α5, β1, RCP or EGFR1 or with truncated GFP-RCP^379-649 as indicated. Cells were serum starved overnight followed by a 40-min treatment with either 1 μM cilengitide or 0.5 μg/ml osteopontin. The cells were then challenged with EGF for the indicated times and lysed. Levels of active phosphoTyr⁸⁴⁵-EGFR1, phosphoSer⁴⁷³-Akt, and phosphoThr²⁰²/phosphoTyr²⁰⁴-ERK were determined by Western blotting. Protein loading of β-actin, Akt1, and EGFR1 was confirmed by Western blotting.