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First published online 8 July 2008
doi: 10.1242/jcs.033001

Journal of Cell Science 121, 2452-2462 (2008)
Published by The Company of Biologists 2008
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Binding of soluble fibronectin to integrin {alpha}5β1 – link to focal adhesion redistribution and contractile shape

Stephan Huveneers1,2, Hoa Truong1, Reinhard Fässler3, Arnoud Sonnenberg2 and Erik H. J. Danen1,2,*

1 Division of Toxicology, Leiden Amsterdam Center for Drug Research, Einsteinweg 55, Leiden University, Leiden 2333 CC, The Netherlands
2 Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands
3 Department of Molecular Medicine, Max Planck Institute of Biochemistry, Am Klopferspitz 18, Martinsried, 82152, Germany


Figure 1
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  		Fig. 1. Integrin β3 is dispensable for RhoA activity and contractility. (A) Schematic representation of previous results. Itgb1-null cells (GE11) have very low levels of GTP-bound RhoA and fibronectin matrix assembly. Re-expression of β1 integrin, but not overexpression of β3 integrin induces highly dynamic cell-matrix adhesions, a mesenchymal cell morphology and restores RhoA activity and fibronectin matrix assembly. Images show GEβ1 and GEβ3 cells stained for paxillin (green) and F-actin (red). (B) Western blot analysis of RhoA activity assay on lysates of Itgb3 knockout and wild-type MEFs overexpressing indicated integrins. Quantification shows relative RhoA activation ± s.d. compared with wild-type MEFs from two independent experiments. (C) Images of assembled fibronectin-biotin on Itgb3-knockout and wild-type MEFs; fibronectin is red and nucleus blue in merged images. Scale bar: 50 µm. (D) Itgb3-knockout and wild-type MEFs overexpressing indicated integrins stained for paxillin (green), F-actin (red). Nuclei are stained blue with Topro-3. Arrows indicate long actin fibers. Scale bar: 10 µm. (E) Flow cytometry analysis of integrin β1 surface expression on Itgb3 knockout and wild-type MEFs transfected with integrin β1 or control siRNA. (F) Wild-type and Itgb3-knockout MEFs transfected with integrin β1 or control siRNA stained for paxillin (green) and F-actin (red). Two representative examples of integrin β1 siRNA transfected wild-type MEFs are shown.

 

Figure 2
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  		Fig. 2. Integrin {alpha}5 is required for efficient RhoA activation and fibronectin fibrillogenesis. (A) Western blot analysis of RhoA activity assay on lysates of EA5 cells expressing indicated integrins. Quantification shows relative RhoA activation ± s.d. compared with EA5 cells from two independent experiments. (B) EA5 cells expressing indicated constructs stained for paxillin (green) and F-actin (red), with the nucleus in blue. Scale bar: 10 µm. (C) Western blot analysis of assembled fibronectin-biotin and vimentin (loading control) in DOC-insoluble lysates of EA5 cells expressing indicated constructs. Locations of molecular size markers (in kDa) are indicated.

 

Figure 3
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  		Fig. 3. Ligand binding to the extracellular domain of β1 integrin is required for RhoA activation and fibronectin fibrillogenesis. (A) GE11 cells expressing indicated β1 integrin subunits stained for paxillin (green), F-actin (red). Nuclei are stained blue with Topro-3. Scale bar: 10 µm. (B) Western blot analysis of RhoA activity assay on lysates of GE11 cells expressing indicated β1 integrin subunits. Quantification shows relative RhoA activation ± s.d. compared with GEβ1 cells from two experiments. (C) Assembled fibronectin-biotin on GE11 cells expressing indicated β1 integrin subunits; fibronectin is red and nuclei are blue. Scale bar: 50 µm. (D) Mean fluorescence analyzed by flow cytometry, demonstrating binding of indicated integrins to different concentrations of soluble FITC-fibronectin.

 

Figure 4
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  		Fig. 4. High-affinity mutants of β3 integrin fail to stimulate RhoA activity and fibronectin fibrillogenesis. (A) Images of β1-integrin-deficient GE11 cells expressing indicated β3 integrin affinity mutants stained for paxillin (green), F-actin (red) and the nucleus (blue). Scale bar: 10 µm. (B) Western blot analysis of RhoA activity assay on lysates of GE11 cells expressing indicated constructs. Quantification shows relative RhoA activation ± s.d. compared with GEβ1 cells from two experiments. (C) Western blot analysis of assembled fibronectin-biotin and vimentin (loading control) in DOC-insoluble lysates of GE11 cells expressing indicated constructs.

 

Figure 5
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  		Fig. 5. Integrin binding of soluble GRGDSP and fibronectin. (A) Mean fluorescence ± s.d. demonstrating GRGDSP-biotin binding (10 µM) to indicated integrins expressed on GE11 cells analyzed by flow cytometry. (B) Mean fluorescence analyzed by flow cytometry, demonstrating binding of indicated integrins to different concentrations of soluble FITC-fibronectin. Binding to integrin β3 was shown previously in Fig. 3D. (C) Mean fluorescence demonstrating binding of soluble fibronectin-biotin (10 µg/ml) to indicated integrins upon competition with increasing concentrations of unlabeled fibronectin analyzed by flow cytometry.

 

Figure 6
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  		Fig. 6. High-affinity binding to the hypervariable region of the β1 I-like domain controls signaling to fibronectin fibrillogenesis. (A) Mean fluorescence analyzed by flow cytometry, demonstrating binding of integrin β1 and β1-3-1 to different concentrations of soluble FITC-fibronectin. (B) Images of assembled fibronectin-biotin on GE11 cells expressing β1 or β1-3-1; fibronectin is red and nucleus blue. Scale bar: 50 µm. (C) Western blot analysis of RhoA activity assay on lysates of GE11 cells expressing indicated integrins. Quantification shows relative RhoA activation ± s.d. compared with GEβ1 cells from two independent experiments. (D) Images of GE11 cells expressing β1 integrin or β1-3-1 stained for paxillin (green) and F-actin (red). Scale bar: 10 µm.

 

Figure 7
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  		Fig. 7. Soluble fibronectin binding to {alpha}5β1 integrin supports Rho GTP-loading independent of ROCK or myosin-II activity or fibronectin fibrillogenesis. (A) Images of assembled fibronectin-biotin on GEβ1 cells treated with inhibitors of ROCK (Y-27632, 10 µM) or myosin-II (Blebbistatin, 50 µM) or with DMSO (control); fibronectin is stained red and nucleus blue. Scale bar: 50 µm. (B) Western blot analysis of RhoA activity assay on lysates of GE11 cells treated with the indicated inhibitors. Quantification shows relative RhoA activation ± s.d. compared with GEβ1 cells from three independent experiments. (C) Binding of soluble fibronectin-biotin (10 µg/ml) to GEβ1 cells treated with the indicated inhibitors.

 

Figure 8
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  		Fig. 8. Syndecan-4 is not required for {alpha}5β1-integrin-supported RhoA signaling. (A) Flow cytometry analysis of endogenous syndecan-4 surface expression on GEβ1 cells transfected with syndecan-4 or control siRNA. (B) Images of GEβ1 cells transfected with syndecan-4 or control siRNA stained for paxillin (green), F-actin (red) and the nucleus (blue). (C) Flow cytometry analysis of soluble FITC-fibronectin (10 µg/ml) binding to GEβ1 cells with or without syndecan-4 knockdown. (D) Images of assembled fibronectin-biotin on GEβ1 transfected with syndecan-4 or control siRNA; fibronectin is red and nucleus is blue. Scale bar: 50 µm. (E) Western blot analysis of RhoA activity assay on lysates of syndecan-4 knockdown and control GEβ1 cells. (F) Flow cytometry analysis of endogenous syndecan-4 surface expression on GEβ3 cells transfected with syndecan-4 or control siRNA. (G) Images of GEβ3 cells transfected with syndecan-4 or control siRNA stained for paxillin (green) and F-actin (red).

 

Figure 9
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  		Fig. 9. A model for the role of {alpha}5β1 integrin in reorganization of focal adhesions, contractility, cell morphology and fibronectin-matrix assembly. (A) Adhesion to immobilized (stretched) RGD-containing ECM components such as vitronectin (VN) or fibronectin (FN) through {alpha}vβ3 integrin in the absence of {alpha}5β1 integrin promotes the formation of randomly distributed cell-matrix adhesions leading to an epithelial-like, flat, circular morphology. (B) In the presence of {alpha}5β1 integrin, binding of compact soluble fibronectin dimers to this integrin promotes the dynamic centripetal redistribution of focal adhesions. Together with the high activity of RhoA that is supported in the presence of {alpha}5β1 integrin, this allows the cytoskeletal organization that drives fibronectin fibrillogenesis.

 

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© The Company of Biologists Ltd 2008