Physical state of the extracellular matrix regulates the structure and molecular composition of cell-matrix adhesions

Abstract

This study establishes that the physical state of the extracellular matrix can regulate integrin-mediated cytoskeletal assembly and tyrosine phosphorylation to generate two distinct types of cell-matrix adhesions. In primary fibroblasts, alpha(5)beta(1) integrin associates mainly with fibronectin fibrils and forms adhesions structurally distinct from focal contacts, independent of actomyosin-mediated cell contractility. These "fibrillar adhesions" are enriched in tensin, but contain low levels of the typical focal contact components paxillin, vinculin, and tyrosine-phosphorylated proteins. However, when the fibronectin is covalently linked to the substrate, alpha(5)beta(1) integrin forms highly tyrosine-phosphorylated, "classical" focal contacts containing high levels of paxillin and vinculin. These experiments indicate that the physical state of the matrix, not just its molecular composition, is a critical factor in defining cytoskeletal organization and phosphorylation at adhesion sites. We propose that molecular organization of adhesion sites is controlled by at least two mechanisms: 1) specific integrins associate with their ligands in transmembrane complexes with appropriate cytoplasmic anchor proteins (e.g., fibronectin-alpha(5)beta(1) integrin-tensin complexes), and 2) physical properties (e.g., rigidity) of the extracellular matrix regulate local tension at adhesion sites and activate local tyrosine phosphorylation, recruiting a variety of plaque molecules to these sites. These mechanisms generate structurally and functionally distinct types of matrix adhesions in fibroblasts.

Figure 1

Distribution of fibronectin, paxillin and tensin in primary human fibroblasts. Cells were cultured for 16 h on coverslips. Double immunofluorescence staining was then performed with antibodies to paxillin (A) and fibronectin (B) or with tensin (C) and fibronectin (D). Note the localization of paxillin and tensin in FCs (arrows) and tensin along fibronectin fibrils (arrowheads). Bar, 20 μm.

Figure 2

Ratio imaging of α₅ integrin with other components of cell-matrix adhesions. Primary human fibroblasts were cultured on coverslips for 16 h. Double immunofluorescence staining was then performed with antibodies to α₅ integrin compared with α_v integrin (mouse anti-α_v integrin monoclonal antibody followed by Cy3-conjugated goat anti-mouse antibody, and rat anti-α₅ integrin monoclonal antibody followed by FITC-conjugated goat anti-rat antibody; first column of panels, left), tensin (mouse anti-tensin monoclonal antibody followed by Cy3-conjugated goat anti-mouse antibody with rat anti-α₅ integrin monoclonal antibody followed by FITC-conjugated goat anti-rat antibody; second column), vinculin (rabbit anti-vinculin followed by FITC-conjugated goat anti-rabbit antibody with rat anti-α₅ integrin monoclonal antibody followed by Cy3-cojugated goat anti-rat antibody; third column), or fibronectin (rat anti-α₅ integrin monoclonal antibody followed by Cy3-cojugated goat anti-rat antibody with FITC-conjugated goat anti-fibronectin antibody; right column). Cy3 and FITC images are presented in the top and second row, respectively. Superimposed images of Cy3 and FITC double-labeled images are shown in the third row. Ratio image analyses of the Cy3 and FITC-double labeled images were performed as described in MATERIALS AND METHODS, and the resulting images are shown in the bottom row. Spectrum color scale indicates the value of the ratios. Note the colocalization of α₅ integrin with tensin and fibronectin in fibrillar adhesions and the sorting of α₅ integrin from α_v integrin and vinculin into distinct structures, as reflected by the ratio images of these components.

Figure 3

F-actin localization at cell-matrix adhesions. Primary human fibroblasts were cultured on coverslips for 16 h. Double immunofluorescence staining was then performed for (A) F-actin (rhodamine-conjugated phalloidin) and (B) α₅ integrin (rat anti-α₅ integrin monoclonal antibody followed by FITC-conjugated goat anti-rat antibody). (A′) and (B′) High-power magnification of the regions defined by rectangles in (A) and (B), respectively. Arrowheads indicate the location of fibrillar adhesions and thin actin filaments. Bar, 15 μm.

Figure 4

Ratio imaging of tyrosine phosphorylation in cell-matrix adhesions. Primary human fibroblasts were cultured on coverslips for 16 h. Double immunofluorescence staining was then performed with antibodies to pTyr with α₅ integrin (rat anti-α₅ integrin monoclonal antibody followed by Cy3-conjugated goat anti-rat antibody compared with mouse anti-pTyr monoclonal antibody followed by FITC-conjugated goat anti-mouse antibody; first column of panels, left), α_v integrin (mouse anti-α_v integrin monoclonal antibody followed by FITC-conjugated goat anti-mouse antibody paired with rabbit anti-pTyr antibody followed by Cy3-cojugated goat anti-rabbit antibody; second column), or fibronectin (rabbit anti-pTyr antibody followed by Cy3-cojugated goat anti-rabbit antibody with FITC-conjugated goat anti-fibronectin antibody; right column). Cy3 and FITC-labeled images are presented in the top and second row, respectively. Superimposed images of Cy3 and FITC-double labeled images are shown in the third row. Ratio image analysis of the Cy3 and FITC-double labeled images was performed as described in MATERIALS AND METHODS, and the resulting images are shown in the bottom row. Spectrum color scale indicates the value of the ratios. Note the colocalization of α_v integrin with pTyr in FCs, whereas the relative amounts of pTyr in fibrillar adhesions (containing α₅ integrin and fibronectin) are low, as reflected by the ratio images of these components.

Figure 5

Effects of covalent immobilization on the quantity and epitope exposure of fibronectin and on cell spreading. Coverslips coated with immobilized or nonimmobilized (control) fibronectin were fixed and immunolabeled for fibronectin using monoclonal antibodies 11E5 or 16G3 or the polyclonal antibody R745. (A) Diagram of the 37-kDa fibronectin cell-binding domain showing locations of the epitopes of monoclonal antibodies 11E5 and 16G3 compared with the RGD site. (B) Bar graph showing the average immunofluorescence labeling in arbitrary units (A.U.) of immobilized or nonimmobilized (control) fibronectin. Primary human fibroblasts were plated for 16 h on control, nonimmobilized fibronectin (C) or immobilized fibronectin as described in MATERIALS AND METHODS (D). Immunofluorescence staining was then performed for fibronectin (FITC-conjugated goat anti-fibronectin; C and D). Note that cells plated on immobilized fibronectin did not form fibrillar adhesions.

Figure 6

α₅β₁ integrin localizes to FCs of cells cultured on immobilized fibronectin. Primary human fibroblasts were cultured for 16 h on control, nonimmobilized fibronectin (A, C, and E) or on immobilized fibronectin (B, D, and F). Immunofluorescence staining was then performed for α₅ integrin (rat anti-α₅ integrin monoclonal antibody followed by FITC-conjugated goat anti-rat antibody; A and B), the total population of β₁ integrin molecules (rat anti-β₁ integrin monoclonal antibody followed by FITC-conjugated goat anti-rat antibody; C and D), or activated β₁ integrin (β₁*) molecules (mouse anti-activated β₁ integrin monoclonal antibody followed by Cy3-conjugated goat anti-mouse antibody; E and F). Activated α₅β₁ integrins localize predominantly in fibrillar adhesions of cells cultured on control fibronectin (arrows), but in the FCs of cells plated on immobilized fibronectin (arrowheads). Bar, 15 μm.

Figure 7

The molecular composition of FCs of cells plated on immobilized fibronectin in the presence of anti-α_v integrin inhibitory antibody. Primary human fibroblasts were cultured on control, nonimmobilized fibronectin (left) or on immobilized fibronectin (right) for 16 h, both in the presence of inhibitory anti-α_v integrin monoclonal antibody. The following immunofluorescence stainings were then performed: α₅ integrin (rat anti-α₅ integrin monoclonal antibody followed by FITC-conjugated goat anti-rat antibody; top row), α_v integrin (mouse anti-α_v integrin monoclonal antibody followed by Cy3-conjugated goat anti-mouse antibody; second row), vinculin (rabbit anti-vinculin antibody followed by Cy3-conjugated goat anti-rabbit antibody; third row), pTyr (rabbit anti-pTyr antibody followed by Cy3-conjugated goat anti-rabbit antibody; fourth row), and F-actin (rhodamine-conjugated phalloidin; bottom row). Bar, 10 μm.

Figure 8

Migration rates are reduced when cells are plated on immobilized fibronectin compared with cells plated on control, nonimmobilized fibronectin. Primary human fibroblasts were plated on coverslips coated with fibronectin substrates as described in MATERIALS AND METHODS. After 16 h, the migration of single cells was recorded for 5 h. Eighteen to 22 cells were examined in each experiment, and each experiment was performed two times. The graph shows the average and SD of data pooled from two independent experiments, and the difference between the migration rates of cells plated on the different substrates is statistically significant (p < 0.0001).

Figure 9

Summary diagram: integrin-mediated cytoskeletal assembly and cell migration are both controlled by the physical status of the ECM. Binding of the α₅β₁ integrin to its ligand fibronectin results in the formation of fibrillar adhesions that contain tensin as a major cytoskeletal component and that are associated with actin filaments. Matrix immobilization prevents matrix reorganization and formation of fibrillar adhesions. When the same integrin binds to an immobilized fibronectin ligand, it forms typical, highly phosphorylated FCs that associate with the termini of actin stress fibers, accompanied by reduction of cell migration rates. The latter FC type of adhesion depends on actomyosin-mediated contractility that can be inhibited by H-7 or ML-7. In contrast, fibrillar adhesions are not disrupted by these inhibitors.