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Proc Natl Acad Sci U S A. 2009 Sep 22; 106(38): 16245–16250.
Published online 2009 Sep 3. doi:  10.1073/pnas.0902818106
PMCID: PMC2752568
Biophysics and Computational Biology

Clustering of α5β1 integrins determines adhesion strength whereas αvβ3 and talin enable mechanotransduction

Abstract

A key molecular link between cells and the extracellular matrix is the binding between fibronectin and integrins α5β1 and αvβ3. However, the roles of these different integrins in establishing adhesion remain unclear. We tested the adhesion strength of fibronectin-integrin-cytoskeleton linkages by applying physiological nanonewton forces to fibronectin-coated magnetic beads bound to cells. We report that the clustering of fibronectin domains within 40 nm led to integrin α5β1 recruitment, and increased the ability to sustain force by over six-fold. This force was supported by α5β1 integrin clusters. Importantly, we did not detect a role of either integrin αvβ3 or talin 1 or 2 in maintaining adhesion strength. Instead, these molecules enabled the connection to the cytoskeleton and reinforcement in response to an applied force. Thus, high matrix forces are primarily supported by clustered α5β1 integrins, while less stable links to αvβ3 integrins initiate mechanotransduction, resulting in reinforcement of integrin-cytoskeleton linkages through talin-dependent bonds.

An important link of a cell with its environment occurs between the extracellular matrix protein fibronectin and its ligands, integrins α5β1 and αvβ3. Both integrins bind to fibronectin mainly through the RGD (Arg-Gly-Asp) and PHSRN (Pro-His-Ser-Arg-Asn) peptide sequences, are present at fibronectin adhesion sites, and enable attachment to fibronectin matrices (1). Cell-fibronectin binding is increased by fibronectin clustering (27) and integrin binding to talin (8, 9). However, whether the two integrins have different functions, and how fibronectin clustering and talin affect these functions, remains thus far unknown.

The functions of integrins in cell-matrix adhesion can be divided in three mechanochemical steps. First, fibronectin-integrin bonds must cooperate to withstand the high (nN) forces (10, 11) present at adhesion sites. Second, these forces have to be translated into biochemical signals (mechanotransduction). Finally, integrins must mechanically connect to the cytoskeleton to transmit forces throughout the cell, enabling an integrated cell response. We thus set out to analyze how integrins α5β1 and αvβ3 and their binding to fibronectin and talin regulate these steps.

To address this issue, we developed a magnetic tweezers apparatus (12) able to exert forces of 1 nN on 2.8-μm diameter magnetic beads coated with FN7–10 (7), a four-domain segment of fibronectin responsible for cell binding and containing the RGD and PHSRN motifs. As mouse embryonic fibroblasts (MEF) spread on glass surfaces coated with laminin (with receptors independent of α5β1 and αvβ3 integrins), they formed adhesions to previously deposited fibronectin beads, picked them up, and started transporting them with the rearward moving actin cytoskeleton. After observing the rearward flow for 20 s, a pulsatile 1 nN force directed toward the cell edge was exerted on the beads (Fig. 1 A–C and Movie S1) for 100 s. The forces applied enabled us to separately study the different steps involved. First, these forces were high enough to detach a fraction of the beads. This allowed us to measure bead-cell adhesive strength as the percentage of beads still attached to the cell after force application (Movie S2). Second, mechanotransduction was assessed as the ability of cells to sense force and reinforce adhesion sites (13) by decreasing force-dependent bead pulsation over time (Fig. 1 D and E). Finally, linkage to the cytoskeleton was evaluated by measuring bead rearward velocity relative to actin flow velocity before force application (Fig. 1D).

Fig. 1.
Experimental setup. (A) Diagram showing the magnetic tweezers apparatus. A current (I, white arrow) goes through a set of coils placed around a magnetic core, which creates a magnetic gradient around the core tip. The force exerted on the bead (Fbead ...

Results

We first analyzed how fibronectin clustering regulated adhesion strength by coating beads with either monomeric FN7–10 or with a pentameric form (Fig. 2A), in which 5 FN7–10 molecules were clustered within approximately 40 nm (7). By coating with a mixture of FN7–10 and different concentrations of BSA, we regulated the density of fibronectin on the beads, with higher BSA concentrations displacing fibronectin and resulting in less fibronectin bound to beads (Fig. 2B). The amount of fibronectin per bead in each case was obtained by gel densitometry, calibrated with known concentrations of FN7–10 (Fig. 2C). From this quantity, we obtained the average force exerted per individual FN7–10 subunit (Fig. 2D) using the total force of 1 nN per bead and the average bead-cell contact area. This area was determined by α5β1 integrin staining in confocal sections and ranged from 50–60% of bead surface independent of fibronectin concentration or multimerization (Fig. S1). Whereas beads coated only with BSA had 0% attachment, the percentage of pentamer-coated beads that remained attached after force was systematically higher than that of monomer-coated beads (Fig. 2D). Further, beads coated with pentamer had a tendency to release slower (Fig. S2 A and B). After assuming a sigmoid dependence of the percentage of adhered beads on ligand concentration (lines on Fig. 2D, see statistical analysis in Fig. S2C) and taking 50% attachment as a tipping point between stable and unstable adhesion, the maximum average force per FN7–10 molecule for stable adhesion was approximately 0.65 pN for pentamer-coated beads, but only approximately 0.1 pN for monomer-coated beads. These values are an average measure of adhesion forces per molecule, since several parameters (small differences in applied force, variability in bead embedding, angle of force application) could affect the force applied to individual molecules. However, these parameters did not depend on bead coating with monomeric versus pentameric FN7–10. Thus, the clustering of fibronectin (and therefore of integrin ligands) increased six-fold the force adhesion sites could withstand.

Fig. 2.
Clustered fibronectin receptors withhold force over six-fold more efficiently than non-clustered ones. (A) Scheme of monomeric and pentameric FN7–10 constructs with fibronectin (fn) domains, tenascin (tn) spacer domains, and COMP pentamerization ...

Exposure to soluble linear RGD peptides, which bind to both major fibronectin-binding integrins α5β1 and αvβ3, inhibits cell adhesion to fibronectin (14). We thus investigated which of these two major integrins supported high forces. After force application, most pentamer-coated beads remained attached in control cells, while in cells treated with an inhibitory antibody against α5β1 integrin all beads detached (Fig. 3 A–C). In contrast, cells treated with 0.5 mM of the cyclic GPenRGDSPCA (GPen) peptide [a concentration which inhibited binding of fibronectin to αvβ3 but not to α5β1 (14)], there was no effect on bead adhesion (Fig. 3 A–C). However, 0.5 mM GPen peptide blocked cell spreading on surfaces coated with vitronectin, a major αvβ3 ligand (Fig. S3). Further, beads on cells treated with α5β1 antibody, but not with GPen peptide, had decreased detachment times (Fig. 3C) and increased diffusion (mean squared displacement perpendicular to cell rearward flow), signifying weaker adhesion (Fig. 3D). Therefore, integrin α5β1 and not αvβ3 was responsible for adhesion strength to fibronectin.

Fig. 3.
High force resistance is provided by ligation of α5β1 but not αvβ3 integrins. (A) Examples of cells (control and exposed to 10 μg/mL α5β1 antibody or 0.5 mM of GPen peptide) with attached beads coated ...

Even though αvβ3 integrin did not maintain adhesion strength under force, we previously reported that αvβ3 inhibition blocked the cell's ability to maintain the rearward movement of fibronectin-coated beads after applying small forces (15). We thus hypothesized that αvβ3 might reinforce adhesion sites by transducing force into a stiffening signal rather than by increasing adhesion strength. To test this, we analyzed bead movement in response to pulsatory force after treatment with GPen. Beads in control cells reduced their amplitude of movement with time (Fig. 4A), and stiffened (Fig. 4B). This stiffening was not affected by possible changes in focus during bead rearward transport (Fig. S4), nor by changes in force caused by varying tip-bead distances, as cell stiffness at each time point was calculated from the force applied at the same time point. Therefore, stiffening was due to adhesion site reinforcement. However, beads in GPen treated cells did not reinforce (Fig. 4 A and B). In a similar manner, β3-null cells did not reinforce either, but had normal adhesion strength (Fig. S5). Thus, αvβ3 was necessary to transduce force into a stiffening response, but not to maintain adhesion strength.

Fig. 4.
Integrin αvβ3 and talin enable mechanotransduction. (A) Sample traces of displacement in the direction of force exertion as a function of time for beads coated with pentameric FN7–10 on control cells (black) and cells treated with ...

We then analyzed whether talin, which binds both integrins α5β1 and αvβ3 (8, 9) and actin (16) and has a role in adhesion sites (6, 15, 17), regulated adhesion strength or reinforcement. While MEF talin1−/− cells showed normal morphology due to talin2 overexpression (18), transfection of a talin2-siRNA plasmid [which reduced talin expression by 48–68% (18)] resulted in a rounded cell shape in culture after 3 days, but did not abolish the initial spreading phase, as previously described (18), nor the ability of cells to pick up and transport beads. After talin depletion, cells lost the stiffening response (Fig. 4 C and D). This response was restored by transfecting GFP-talin1 (Fig. 4 C and D). However, talin depletion did not significantly affect the percentage of beads that remained attached after force application (Fig. S6). Thus, talin was involved in mechanotransduction and stiffening of bead-cytoskeleton linkages but not in maintaining adhesion strength.

We next examined whether integrins and talin also formed a mechanical connection to the actin cytoskeleton. To test this, we measured the rearward movement (toward the cell center) of fibronectin coated beads not subjected to force (Fig. 5 A and C) and of cytoplasmic markers (Fig. 5 A and E). Cytoplasmic markers were indicators of fibroblast actin rearward flow (19). By comparing both quantities, we determined whether the fibronectin-integrin adhesions formed around beads coupled to the rearward moving cytoskeleton. When either α5β1 or αvβ3 integrins were inhibited, the rearward bead velocity was halved, while cytoplasmic marker movements were unaffected (Fig. 5G). Thus, while cytoskeletal rearward flow remained unchanged, the coupling of beads to this flow (bead speed/cytoplasmic speed) was reduced from approximately 80% in control cells to approximately 30% after integrin inhibition. Due to the very slow actin rearward flow in β3+/+ and β3−/− cells, however, differences could not be observed between these cell lines. As a positive control, we tracked the rearward movement of beads and cytoplasmic markers in Myosin IIA knock-down cells and cells treated with blebbistatin, which inhibits myosin II function and actin rearward flow (20). In both cases, myosin II inhibition decreased the rearward velocity of bead and actin markers in parallel (Fig. S7). Beads coated with monomeric instead of pentameric FN7–10 also had reduced velocities (30% of actin markers) (Fig. 5), indicating that integrins must aggregate while bound to clustered ligands to connect to the cytoskeleton.

Fig. 5.
Binding of both integrins to clustered fibronectin receptors and recognition of these clusters by talin is required to mechanically connect to the actin cytoskeleton. (A) Images of control MEF cells and cells treated with 10 μg/mL of inhibitory ...

Talin was also necessary for cytoskeletal connection, since talin1−/− cells (with and without talin2 siRNA transfection) had low rearward speeds and a weak coupling to cytoskeletal flow of only approximately 30%. Co-transfection of talin2 siRNA and talin1 GFP restored rearward velocity and cytoskeletal coupling to 80% (Fig. 5 B, D, F, and H). Thus, although they reinforced, talin1−/− cells did not exhibit a normal cytoskeletal connection, which required talin1-GFP expression. This might indicate that talin 1 and 2 have different roles. However, it could simply indicate that talin 2 overexpression in talin1−/− cells (18) is enough to enable reinforcement but not to rescue rearward movement, which might require a higher talin concentration to maintain a thorough integrin-cytoskeleton mechanical connection. Finally, the restoration of cytoskeletal coupling induced by talin1-GFP expression did not occur with monomeric FN7–10 (Fig. 5H), showing that talin must bind to clustered integrins to mechanically connect to the actin cytoskeleton.

Given the important role of integrin clustering in focal adhesion formation, we finally analyzed whether fibronectin clustering affected integrin and talin recruitment. Whereas beads attached in the perinuclear region showed strong recruitment of both integrins and talin, beads still traveling through the lamellipodium (the zone used for force measurements) were more likely to show α5β1 and talin1 recruitment if coated with pentameric FN7–10 (Fig. 6). This recruitment was due to increased integrin and talin localization and not merely to bead embedding, as shown by vertical confocal slices (Fig. 6B). This was under conditions where bead FN7–10 concentration was 4-fold higher in monomer-coated beads than in pentamer-coated beads (1:10 fibronectin:BSA coating used in both cases, see Fig. 2B). Therefore, clustering of fibronectin receptors, even if their total concentration is lower, induces earlier recruitment and clustering of integrins and talin.

Fig. 6.
Fibronectin clustering induces integrin and talin recruitment. (A) Left panel: DIC images of MEF cells with pentameric and monomeric-FN7–10 coated beads. Superimposed red labeling differentiates between coatings. Right panels: corresponding epifluorescence ...

Discussion

In our previous work focused on the reinforcement step of cell adhesion (6, 21, 22) (for small pN forces, small surface area and a timescale of seconds) we reported a strong dependence upon integrin αvβ3 and talin. Here we found that the three different functions of 1) high force adhesion, 2) reinforcement, and 3) linkage to the cytoskeleton depended upon different molecular complexes. While fibronectin clustering and integrin α5β1 determined adhesion strength, integrin αvβ3 and talin enabled reinforcement and mechanotransduction, with talin also recognizing integrin clusters and linking them to the cytoskeleton. Thus, we suggest that there is a different molecular regulation of the three mechanochemical steps of adhesion formation.

Our results lead to the intriguing but intuitive idea that integrins might have somewhat opposing mechanical roles. While a stable adhesion requires a strong molecular bond to resist high forces (provided by integrin α5β1), mechanotransduction might entail a weaker bond able to facilitate force detection by breaking more easily. This function would be provided by integrin αvβ3, which could not support high forces when α5β1 was inhibited. Earlier measurements of single fibronectin-α5β1 bond strength are on the order of tens of pN (23, 24), but we observed bead detachment with much lower average forces per FN7–10 molecule (0.1–0.65 pN). We suggest that cells are not able to apply force equally or bind to all fibronectin ligands presented on the beads. Thus, individual molecules may sustain higher forces. This high adhesion strength of single fibronectin-α5β1 bonds, combined with the recent finding that α5β1–fibronectin links form catch bonds that strengthen under force (25, 26), also support our finding that α5β1 maintains adhesion strength. Conversely, our observation that αvβ3 is unable to maintain adhesion by itself indicates that bonds to αvβ3 should break easily under the forces reported here, although they could subsequently rebind. Binding of integrins to extracellular matrix proteins induces downstream signaling such as activation of Src-family kinases through activation of RPTPα (22) or PTP-1B (27). Thus, a fast rate of binding/unbinding of αvβ3 might provide a mechanism for continuous force sensing. In support of this hypothesis, Src colocalizes with αv but not β1 integrins (which are markers respectively of αvβ3 and α5β1) (28). Force sensing could then result in stiffening through recruitment of additional integrins or other cytoskeletal proteins.

Our results on the roles of α5β1 and αvβ3 integrins help interpret previous findings. α5β1 integrins colocalize initially with αvβ3 integrins in focal contacts at the cell edge, but subsequently translocate toward the cell interior (29, 30). The cell leading edge is highly dynamic (31) and is believed to function as a probe of the cell mechanical environment (32). Thus, this is an ideal location for the mechanosensory function of αvβ3 integrins, exemplified by the fact that their recycling to focal contacts is required for persistent migration (33). Conversely, more interior zones where α5β1 predominates are less dynamic (31), α5β1 and not αvβ3 regulates fibrillogenesis (34), and increasing α5β1 versus αvβ3 recycling stops migration (33), supporting a role of α5β1 in establishing stronger structural adhesions.

Clustering of FN7–10 domains was also essential for strong adhesion. Indeed, at the 50% adhesion point determined from sigmoidal fits, the corresponding average spacing between FN7–10 molecules is of approximately 50 nm for monomer-coated beads [thus at the previously established threshold for cell adhesion (2)] but of approximately 130 nm for pentamer-coated beads (Fig. S2D). Thus, local clusters of FN7–10 molecules enhance adhesion, even if average spacing between molecules is higher. There are several possible explanations for the dependence of adhesion strength on FN7–10 clustering. Two logical possibilities are that 1) the proximity of the integrin cytoplasmic domains enables the recruitment of a stabilizing protein complex which increases collective or individual adhesion strength or 2) fibronectin clustering increases lateral integrin interactions (35), promoting α5β1 integrin recruitment as observed (Fig. 6) and increasing the fraction of FN7–10 molecules bound to integrins. In both cases, clustering would also reduce integrin and fibronectin diffusion after bond breakage, facilitating their reattachment and enhancing adhesion. These mechanisms would not require further binding of integrins to talin or the cytoskeleton to permit adhesion, and would therefore explain why talin or myosin II depletion did not affect adhesion strength [Fig. S2 and previously observed (26)] or prevent initial cell spreading (18, 20). From these measurements, it is thus clear that fibronectin fibers or other fibronectin aggregates would have much greater adhesion strength than single molecules.

These studies provide insights into the role of talin in adhesions. Although adhesion strength does not depend on talin, talin is necessary for coupling to the actin rearward flow. Further, while talin is known to induce αvβ3 integrin clustering (36), here we observe that this mechanism operates in both directions. That is, an externally induced integrin clustering also leads to talin binding and recruitment, and this is required to mechanically link the adhesion site to the cytoskeleton and to enable reinforcement. Talin could mediate reinforcement by different mechanisms, such as inducing further integrin recruitment under force. However, our observation that talin mediates both reinforcement and cytoskeletal connection strongly suggests that talin reinforces adhesion sites by increasing the mechanical connection to the cytoskeleton (and thus to the cell actomyosin machinery) under force. Indeed, we recently observed that talin increases its binding to the cytoskeletal protein vinculin when subjected to force (37). Nevertheless, we found that initial spreading on fibronectin is not dependent on talin (18), and we observe here that even though talin is involved in integrin activation (8, 9), its role is mainly that of a scaffold enabling mechanotransduction and linkage to the cytoskeleton, and not an adhesive one. This could be explained by the recent finding (8) that talin-mediated integrin activation is more important with β3 integrins (which had no role in adhesion strength) than with β1 integrins, the major adhesion strength bearers. Even if talin does not support adhesion per se, its enabling of reinforcement could increase long term adhesive strength through adhesion molecule recruitment and focal adhesion maturation (18, 38). This could explain the rounding up of talin1−/− cells treated with talin2 siRNA after initial spreading (18) or the detachment between integrins and actin in talin-null drosophila embryos (38). It must be noted that we cannot rule out the possibility that remaining non-silenced talin2 in talin1−/− cells transfected with talin2 siRNA still contributed to adhesion strength. However, the strong deficiencies of those cells in reinforcement and spreading (18) suggest that any important effect of talin in adhesion strength would have been clearly detectable. Thus, the results that inhibition of talin [and myosin II, Fig. S6 and (26)] do not affect adhesion strength indicate that the molecular links through which cells generate internal forces, responsible for reinforcement and rearward transport, are not necessary to resist external forces. This function would be provided by other mechanisms, which we show to include α5β1 integrins.

We suggest a model for the mechanics of fibronectin-cell contacts in which fibronectin clustering leads to integrin binding, clustering and recruitment. Force applied to the cell is then supported mainly by clustered α5β1 integrins, which can resist forces because broken integrin-fibronectin links quickly rebind due to reduced diffusion of neighboring binding sites. Fibronectin-αvβ3 links are however less stable, and their more frequent force-induced binding/unbinding events might initiate signal transduction, leading to adhesion reinforcement. Finally, binding of talin to integrin clusters mechanically connects the adhesion site to the cytoskeleton and enables reinforcement. Thus, while adhesion strength is mediated mainly by integrin α5β1, reinforcement and mechanotransduction require αvβ3, and talin mechanically connects the adhesion site to the cytoskeleton.

Materials and Methods

Cell Culture.

The MEF and derived Myosin IIA knock-down fibroblast cell lines [previously described (20) as RPTPα+/+] were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). The MEF talin1−/− fibroblast cell line [previously described (18)] was cultured in DMEM/F12 medium supplemented with 15% FBS (all from Gibco).

Bead Preparation.

Carboxylated magnetic beads of 2.8-μm diameter (Invitrogen) were first avidinated by incubating for 2 h 15′ at 0.8% (wt/vol) in a sodium acetate buffer (0.01 M, pH 5.0) with 1.25 mg/mL neutravidin (Invitrogen) and 2.5 mg/mL carbodiimide (Polysciences) at room temperature. Beads were then washed in phosphate buffer saline (PBS), incubated with ethanolamide solution for 30′, washed with PBS again, and resuspended in nonbiotinylated BSA solution (10 mg/mL) in PBS. Avidinated beads were then incubated with 50 μg/mL of biotinylated FN7–10 (in monomeric or pentameric form) and biotinylated BSA (Sigma) for 3 h (at 500 μg/mL unless otherwise specified), washed with PBS, and incubated overnight at 4 °C with 10 mg/mL biotinylated BSA to passivate remaining unbound neutravidin in beads. Beads were then washed in 10 mg/mL BSA in PBS and stored at 4 °C until use. Beads were in continuous rotation in all steps to prevent settling.

Quantification of Bead Functionalization.

To quantify the amount of FN7–10 in beads, beads were prepared as for measurements, mixed with 1× Laemli buffer (Bio-Rad), boiled at 100 °C for 10 min and loaded onto 7.5% polyacrylamide gels (Bio-Rad). A gradient of known concentrations of FN7–10 was prepared and loaded in the same way. Protein was then transferred to an Optitran reinforced nitrocellulose membrane (Whatman), which was blocked with 5% dry milk-Tris buffered saline (TBS) and incubated with mouse anti-fibronectin binding domain antibody (Chemicon) overnight at 4 °C. After incubating for 1 h at room temperature with anti-mouse horseradish peroxidase (Jackson Laboratories), the membrane was blocked with TBS with 0.05% Tween-20 (Fisher Biotech) for 1 h. Marked protein was detected with ECL Western blotting detecting reagents (Amersham Biosciences) on Kodak BioMax XAR film. The signal was quantified using Imagej software, and was converted to protein concentrations using the reference FN7–10 gradient gel lanes.

Force Measurements.

To apply forces to magnetic beads, a previously described magnetic tweezers apparatus (12) was used. Briefly, an electromagnet with a sharpened ferromagnetic core was used to apply a strong magnetic field gradient, generating a force on the beads. The force exerted by the tweezers was calibrated from the velocity of beads in liquids of known viscosity measured as a function of the tip-bead distance and applied current. For force measurements, fibronectin-coated beads were deposited on coverslips silanized with 1,1,1,3,3,3,-hexamethyldisilazane (Aldrich) and coated with 40 μg/mL laminin (Sigma) for 2 h at 37 °C. Coating with laminin (with integrin receptors different to fibronectin) prevented any effect of α5β1 or αvβ3 inhibition on cell spreading. In Fig. S3, coating was done with 10 μg/mL vitronectin (Invitrogen). Cells were then trypsinized, resuspended in Ringer buffer solution (150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, and 2 g/L glucose, pH 7.4) for 30 min at 37 °C for recovery, and plated on the coverslips. The system was then mounted on a motorized 37 °C stage on an Olympus IX81 fluorescence microscope. DIC images and videos were taken with a 60× objective and a Cascade II CCD camera (Photometrics) at a frequency of 12.8 Hz.

Constructs, transfection, antibodies, and chemicals used, and analysis of bead recruitment, bead stiffness and detachment data, and statistics are available as SI Methods.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Y. Cai, X. Zhang, N. Biais, S. Moore, J. Sable, and M. Tanase for technical support and useful discussions, as well as all of the members of the Sheetz laboratory. This work was funded by National Institutes of Health Roadmap for Medical Research Grant PN2 EY016586 (to M.P.S.); Marie Curie International Outgoing Fellowship within the 7th European Community Framework Program Grant PIOF-GA-2008-219401 (to P.R.C.). and a National Institutes of Health award (to A.d.R.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. K.Y. is a guest editor invited by the Editorial Board.

This article contains supporting information online at www.pnas.org/cgi/content/full/0902818106/DCSupplemental.

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