Clustering of �5�1 integrins determines adhesionstrength whereas �v�3 and talinenable mechanotransductionPere Roca-Cusachsa,b, Nils C. Gauthiera, Armando del Rioa, and Michael P. Sheetza,1

aDepartment of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, New York, NY 10027; and bInstitute for Bioengineering of Catalonia,c/Baldiri Reixac 10-12, 08028 Barcelona, Spain

Edited by Kenneth Yamada, National Institutes of Health, Bethesda, MD, and accepted by the Editorial Board July 30, 2009 (received for reviewMarch 13, 2009)

A key molecular link between cells and the extracellular matrix isthe binding between fibronectin and integrins �5�1 and �v�3.However, the roles of these different integrins in establishingadhesion remain unclear. We tested the adhesion strength offibronectin-integrin-cytoskeleton linkages by applying physiolog-ical nanonewton forces to fibronectin-coated magnetic beadsbound to cells. We report that the clustering of fibronectin domainswithin 40 nm led to integrin �5�1 recruitment, and increased theability to sustain force by over six-fold. This force was supported by�5�1 integrin clusters. Importantly, we did not detect a role of eitherintegrin �v�3 or talin 1 or 2 in maintaining adhesion strength. Instead,these molecules enabled the connection to the cytoskeleton andreinforcement in response to an applied force. Thus, high matrixforces are primarily supported by clustered �5�1 integrins, while lessstable links to �v�3 integrins initiate mechanotransduction, resultingin reinforcement of integrin-cytoskeleton linkages through talin-dependent bonds.

An important link of a cell with its environment occursbetween the extracellular matrix protein fibronectin and itsligands, integrins �5�1 and �v�3. Both integrins bind to fibronec-tin mainly through the RGD (Arg-Gly-Asp) and PHSRN (Pro-His-Ser-Arg-Asn) peptide sequences, are present at fibronectinadhesion sites, and enable attachment to fibronectin matrices(1). Cell-fibronectin binding is increased by fibronectin cluster-ing (2–7) and integrin binding to talin (8, 9). However, whetherthe two integrins have different functions, and how fibronectinclustering and talin affect these functions, remains thus farunknown.

The functions of integrins in cell-matrix adhesion can bedivided 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 tobe translated into biochemical signals (mechanotransduction).Finally, integrins must mechanically connect to the cytoskeletonto transmit forces throughout the cell, enabling an integrated cellresponse. We thus set out to analyze how integrins �5�1 and �v�3and their binding to fibronectin and talin regulate these steps.

To address this issue, we developed a magnetic tweezersapparatus (12) able to exert forces of 1 nN on 2.8-�m diametermagnetic beads coated with FN7–10 (7), a four-domain segmentof fibronectin responsible for cell binding and containing theRGD and PHSRN motifs. As mouse embryonic fibroblasts(MEF) spread on glass surfaces coated with laminin (withreceptors independent of �5�1 and �v�3 integrins), they formedadhesions to previously deposited fibronectin beads, pickedthem up, and started transporting them with the rearwardmoving actin cytoskeleton. After observing the rearward flowfor 20 s, a pulsatile 1 nN force directed toward the cell edge wasexerted on the beads (Fig. 1 A–C and Movie S1) for 100 s. Theforces applied enabled us to separately study the different stepsinvolved. First, these forces were high enough to detach afraction of the beads. This allowed us to measure bead-cell

adhesive strength as the percentage of beads still attached to thecell after force application (Movie S2). Second, mechanotrans-duction was assessed as the ability of cells to sense force andreinforce adhesion sites (13) by decreasing force-dependent beadpulsation over time (Fig. 1 D and E). Finally, linkage to thecytoskeleton was evaluated by measuring bead rearward velocityrelative to actin flow velocity before force application (Fig. 1D).

ResultsWe first analyzed how fibronectin clustering regulated adhesionstrength by coating beads with either monomeric FN7–10 or witha pentameric form (Fig. 2A), in which 5 FN7–10 molecules wereclustered within approximately 40 nm (7). By coating with amixture of FN7–10 and different concentrations of BSA, weregulated the density of fibronectin on the beads, with higherBSA concentrations displacing fibronectin and resulting in lessfibronectin bound to beads (Fig. 2B). The amount of fibronectinper bead in each case was obtained by gel densitometry, cali-brated with known concentrations of FN7–10 (Fig. 2C). Fromthis quantity, we obtained the average force exerted per indi-vidual FN7–10 subunit (Fig. 2D) using the total force of 1 nN perbead and the average bead-cell contact area. This area wasdetermined by �5�1 integrin staining in confocal sections andranged from 50–60% of bead surface independent of fibronectinconcentration or multimerization (Fig. S1). Whereas beadscoated only with BSA had 0% attachment, the percentage ofpentamer-coated beads that remained attached after force wassystematically higher than that of monomer-coated beads (Fig.2D). Further, beads coated with pentamer had a tendency torelease slower (Fig. S2 A and B). After assuming a sigmoiddependence of the percentage of adhered beads on ligandconcentration (lines on Fig. 2D, see statistical analysis in Fig.S2C) and taking 50% attachment as a tipping point betweenstable and unstable adhesion, the maximum average force perFN7–10 molecule for stable adhesion was approximately 0.65 pNfor pentamer-coated beads, but only approximately 0.1 pN formonomer-coated beads. These values are an average measure ofadhesion forces per molecule, since several parameters (smalldifferences in applied force, variability in bead embedding, angleof force application) could affect the force applied to individualmolecules. However, these parameters did not depend on beadcoating with monomeric versus pentameric FN7–10. Thus, the

Author contributions: P.R.-C., N.C.G., A.d.R., and M.P.S. designed research; P.R.-C., N.C.G.,and A.d.R. performed research; P.R.-C. contributed new reagents/analytic tools; P.R.-C.analyzed data; and P.R.-C. and M.P.S. wrote the paper.

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.

1To whom correspondence should be addressed at: 713 Fairchild Center, M.C. 2408, NewYork, NY 10027. E-mail: ms2001@columbia.edu.

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

www.pnas.org�cgi�doi�10.1073�pnas.0902818106 PNAS � September 22, 2009 � vol. 106 � no. 38 � 16245–16250

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clustering of fibronectin (and therefore of integrin ligands)increased six-fold the force adhesion sites could withstand.

Exposure to soluble linear RGD peptides, which bind to bothmajor fibronectin-binding integrins �5�1 and �v�3, inhibits celladhesion to fibronectin (14). We thus investigated which of thesetwo major integrins supported high forces. After force applica-tion, most pentamer-coated beads remained attached in controlcells, while in cells treated with an inhibitory antibody against�5�1 integrin all beads detached (Fig. 3 A–C). In contrast, cellstreated with 0.5 mM of the cyclic GPenRGDSPCA (GPen)peptide [a concentration which inhibited binding of fibronectinto �v�3 but not to �5�1 (14)], there was no effect on beadadhesion (Fig. 3 A–C). However, 0.5 mM GPen peptide blockedcell spreading on surfaces coated with vitronectin, a major �v�3ligand (Fig. S3). Further, beads on cells treated with �5�1antibody, but not with GPen peptide, had decreased detachmenttimes (Fig. 3C) and increased diffusion (mean squared displace-ment perpendicular to cell rearward flow), signifying weakeradhesion (Fig. 3D). Therefore, integrin �5�1 and not �v�3 wasresponsible for adhesion strength to fibronectin.

Even though �v�3 integrin did not maintain adhesion strengthunder force, we previously reported that �v�3 inhibition blockedthe cell’s ability to maintain the rearward movement of fibronec-tin-coated beads after applying small forces (15). We thus

hypothesized that �v�3 might reinforce adhesion sites by trans-ducing force into a stiffening signal rather than by increasingadhesion strength. To test this, we analyzed bead movement inresponse to pulsatory force after treatment with GPen. Beads incontrol cells reduced their amplitude of movement with time(Fig. 4A), and stiffened (Fig. 4B). This stiffening was not affectedby 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 forceapplied at the same time point. Therefore, stiffening was due toadhesion site reinforcement. However, beads in GPen treatedcells did not reinforce (Fig. 4 A and B). In a similar manner,�3-null cells did not reinforce either, but had normal adhesionstrength (Fig. S5). Thus, �v�3 was necessary to transduce forceinto a stiffening response, but not to maintain adhesion strength.

We then analyzed whether talin, which binds both integrins�5�1 and �v�3 (8, 9) and actin (16) and has a role in adhesion sites(6, 15, 17), regulated adhesion strength or reinforcement. WhileMEF talin1�/� cells showed normal morphology due to talin2overexpression (18), transfection of a talin2-siRNA plasmid[which reduced talin expression by 48–68% (18)] resulted in arounded cell shape in culture after 3 days, but did not abolish the

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Fig. 1. Experimental setup. (A) Diagram showing the magnetic tweezersapparatus. A current (I, white arrow) goes through a set of coils placed arounda magnetic core, which creates a magnetic gradient around the core tip. Theforce exerted on the bead (Fbead), which increases with this gradient, isstronger as the bead and the magnetic tip get closer. (B) Differential inter-ference contrast (DIC) image showing the magnetic tip, a cell and an attachedmagnetic bead coated with FN7–10. The force exerted on the bead by themagnet pulls the bead toward the cell edge. The graph at the image bottomshows the dependency of applied force on distance to the tip. (Scale bar, 20�m.) (C) Force sequence applied to the measured bead. No force is appliedduring the first 20 s of recording, and then subsequent pulses of 0.5 s offorce/0.5 s without force are applied. Force is calculated from tip-bead dis-tance. (D) Corresponding bead displacement in the direction toward the cellcenter. Before force is applied, beads move toward the cell center and awayfrom the magnetic tip. When force is applied, beads that do not detach startpulsating accordingly and temporarily revert their movement toward the tip.Actual bead movement shown in black, red line is filtered to account for beadpulsation. (E) Subtraction between black and red lines from (D), showing onlythe pulsatory bead response. As time progresses the adhesion around thebead stiffens, decreasing the amplitude of movement.

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initial spreading phase, as previously described (18), nor theability of cells to pick up and transport beads. After talindepletion, cells lost the stiffening response (Fig. 4 C and D). Thisresponse was restored by transfecting GFP-talin1 (Fig. 4 C andD). However, talin depletion did not significantly affect thepercentage of beads that remained attached after force appli-cation (Fig. S6). Thus, talin was involved in mechanotransduc-tion and stiffening of bead-cytoskeleton linkages but not inmaintaining adhesion strength.

We next examined whether integrins and talin also formed amechanical connection to the actin cytoskeleton. To test this, wemeasured the rearward movement (toward the cell center) offibronectin coated beads not subjected to force (Fig. 5 A and C)and of cytoplasmic markers (Fig. 5 A and E). Cytoplasmicmarkers were indicators of fibroblast actin rearward flow (19).By comparing both quantities, we determined whether thefibronectin-integrin adhesions formed around beads coupled to

the rearward moving cytoskeleton. When either �5�1 or �v�3integrins were inhibited, the rearward bead velocity was halved,while cytoplasmic marker movements were unaffected (Fig. 5G).Thus, while cytoskeletal rearward flow remained unchanged, thecoupling of beads to this f low (bead speed/cytoplasmic speed)was reduced from approximately 80% in control cells to approx-imately 30% after integrin inhibition. Due to the very slow actinrearward flow in �3�/� and �3�/� cells, however, differencescould not be observed between these cell lines. As a positivecontrol, we tracked the rearward movement of beads andcytoplasmic markers in Myosin IIA knock-down cells and cellstreated with blebbistatin, which inhibits myosin II function andactin rearward flow (20). In both cases, myosin II inhibitiondecreased the rearward velocity of bead and actin markers inparallel (Fig. S7). Beads coated with monomeric instead ofpentameric FN7–10 also had reduced velocities (30% of actinmarkers) (Fig. 5), indicating that integrins must aggregate whilebound to clustered ligands to connect to the cytoskeleton.

Talin was also necessary for cytoskeletal connection, sincetalin1�/� cells (with and without talin2 siRNA transfection) hadlow rearward speeds and a weak coupling to cytoskeletal f low ofonly approximately 30%. Co-transfection of talin2 siRNA andtalin1 GFP restored rearward velocity and cytoskeletal couplingto 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 thattalin 1 and 2 have different roles. However, it could simplyindicate that talin 2 overexpression in talin1�/� cells (18) isenough to enable reinforcement but not to rescue rearwardmovement, which might require a higher talin concentration tomaintain a thorough integrin-cytoskeleton mechanical connec-

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Fig. 3. High force resistance is provided by ligation of �5�1 but not �v�3integrins. (A) Examples of cells (control and exposed to 10 �g/mL �5�1 anti-body or 0.5 mM of GPen peptide) with attached beads coated with pentamericFN7–10 before and after the application of a pulsatory 1 nN force for 100 s.(Scale bar, 20 �m.) (B) Corresponding traces of bead displacement for controlcells (black line) and cells treated with �5�1 antibody (yellow line) or GPenpeptide (red line). Closed, gray, and open arrows mark respectively thebeginning of the traces, the beginning of force application, and the end of thetrace. In the yellow trace, the straight line depicted when force is appliedrepresents bead detachment from the cell. (C) Percentage of attached beadsafter applying a pulsatory force for 100 s (P � 0.001 for �5�1 compared tocontrol, n � 2 experiments with � six cells per experiment), and average beaddetachment time (P � 0.05 for �5�1 compared to control, n � 12 beads). (D)Mean squared displacement as a function of time in the direction perpendic-ular to the cell center before force application (P � 0.001 for �5�1 inhibitedcells with respect to control, n � 34 beads).

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Roca-Cusachs et al. PNAS � September 22, 2009 � vol. 106 � no. 38 � 16247

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tion. Finally, the restoration of cytoskeletal coupling induced bytalin1-GFP expression did not occur with monomeric FN7–10(Fig. 5H), showing that talin must bind to clustered integrins tomechanically connect to the actin cytoskeleton.

Given the important role of integrin clustering in focal adhe-sion formation, we finally analyzed whether fibronectin cluster-ing affected integrin and talin recruitment. Whereas beadsattached in the perinuclear region showed strong recruitment ofboth integrins and talin, beads still traveling through the lamel-lipodium (the zone used for force measurements) were morelikely to show �5�1 and talin1 recruitment if coated withpentameric FN7–10 (Fig. 6). This recruitment was due toincreased integrin and talin localization and not merely to beadembedding, as shown by vertical confocal slices (Fig. 6B). Thiswas under conditions where bead FN7–10 concentration was4-fold higher in monomer-coated beads than in pentamer-coatedbeads (1:10 fibronectin:BSA coating used in both cases, see Fig.2B). Therefore, clustering of fibronectin receptors, even if theirtotal concentration is lower, induces earlier recruitment andclustering of integrins and talin.

DiscussionIn our previous work focused on the reinforcement step of celladhesion (6, 21, 22) (for small pN forces, small surface area anda timescale of seconds) we reported a strong dependence uponintegrin �v�3 and talin. Here we found that the three differentfunctions of 1) high force adhesion, 2) reinforcement, and 3)linkage to the cytoskeleton depended upon different molecularcomplexes. While fibronectin clustering and integrin �5�1 de-termined adhesion strength, integrin �v�3 and talin enabledreinforcement and mechanotransduction, with talin also recog-nizing integrin clusters and linking them to the cytoskeleton.Thus, we suggest that there is a different molecular regulation ofthe three mechanochemical steps of adhesion formation.

Our results lead to the intriguing but intuitive idea thatintegrins might have somewhat opposing mechanical roles.While a stable adhesion requires a strong molecular bond toresist high forces (provided by integrin �5�1), mechanotrans-duction might entail a weaker bond able to facilitate force

detection by breaking more easily. This function would beprovided by integrin �v�3, which could not support high forceswhen �5�1 was inhibited. Earlier measurements of single fi-bronectin-�5�1 bond strength are on the order of tens of pN (23,24), but we observed bead detachment with much lower averageforces per FN7–10 molecule (0.1–0.65 pN). We suggest that cellsare not able to apply force equally or bind to all fibronectin

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Fig. 5. Binding of both integrins to clustered fibronectin receptors and recognition of these clusters by talin is required to mechanically connect to the actincytoskeleton. (A) Images of control MEF cells and cells treated with 10 �g/mL of inhibitory �5�1 antibody or 0.5 mM GPen peptide. (B) Images of control MEFtalin1�/� cells and cells transfected with indicated plasmids. (Scale bars, 20 �m.) (C and D) Corresponding traces of displacement of beads (pentameric FN7–10coating) marked in yellow in (A and B) during 20 s without force application. (E and F) Kymographs showing the rearward movement of cytoplasmic markers.Kymographs show a vertical line depicting the same line of pixels of an image containing a cytoplasmic marker (shown in yellow in A and B) as it changes withtime. (G and H) Average rearward speed of beads coated with pentameric or monomeric FN7–10 and of cytoplasmic markers in MEF (G) and MEF talin1�/� (H)cells with different treatments. (n � 28 beads, n � 10 cells for kymographs). *, P � 0.05; **, P � 0.01; N.S., No significant differences.

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Fig. 6. Fibronectin clustering induces integrin and talin recruitment. (A) Leftpanel: DIC images of MEF cells with pentameric and monomeric-FN7–10coated beads. Superimposed red labeling differentiates between coatings.Right panels: corresponding epifluorescence staining images of �5�1 anti-body, talin1-GFP, and �3-GFP. Gray and red arrows show positions of unla-beled and labeled beads. (Scale bar, 20 �m.) Gray and red insets show zoomedexamples of monomeric (M) and pentameric (P) bead staining (from corre-sponding square in image). (B) Vertical (x-z) reconstructions from confocalsections displaying increased intensity around beads. (Scale bar, 2 �m.) (C)Percentage of pentamer (black) or monomer (red) coated beads on the cellperiphery with visible recruitment (n � 13 cells). *, P � 0.05; **, P � 0.01, N.S.:No significant differences.

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ligands presented on the beads. Thus, individual molecules maysustain higher forces. This high adhesion strength of singlefibronectin-�5�1 bonds, combined with the recent finding that�5�1–fibronectin links form catch bonds that strengthen underforce (25, 26), also support our finding that �5�1 maintainsadhesion strength. Conversely, our observation that �v�3 isunable to maintain adhesion by itself indicates that bonds to �v�3should break easily under the forces reported here, although theycould subsequently rebind. Binding of integrins to extracellularmatrix proteins induces downstream signaling such as activationof Src-family kinases through activation of RPTP� (22) orPTP-1B (27). Thus, a fast rate of binding/unbinding of �v�3might provide a mechanism for continuous force sensing. Insupport of this hypothesis, Src colocalizes with �v but not �1integrins (which are markers respectively of �v�3 and �5�1) (28).Force sensing could then result in stiffening through recruitmentof additional integrins or other cytoskeletal proteins.

Our results on the roles of �5�1 and �v�3 integrins helpinterpret previous findings. �5�1 integrins colocalize initiallywith �v�3 integrins in focal contacts at the cell edge, butsubsequently translocate toward the cell interior (29, 30). Thecell leading edge is highly dynamic (31) and is believed tofunction as a probe of the cell mechanical environment (32).Thus, this is an ideal location for the mechanosensory functionof �v�3 integrins, exemplified by the fact that their recycling tofocal contacts is required for persistent migration (33). Con-versely, more interior zones where �5�1 predominates are lessdynamic (31), �5�1 and not �v�3 regulates fibrillogenesis (34),and increasing �5�1 versus �v�3 recycling stops migration (33),supporting a role of �5�1 in establishing stronger structuraladhesions.

Clustering of FN7–10 domains was also essential for strongadhesion. Indeed, at the 50% adhesion point determined fromsigmoidal fits, the corresponding average spacing betweenFN7–10 molecules is of approximately 50 nm for monomer-coated beads [thus at the previously established threshold for celladhesion (2)] but of approximately 130 nm for pentamer-coatedbeads (Fig. S2D). Thus, local clusters of FN7–10 moleculesenhance adhesion, even if average spacing between molecules ishigher. There are several possible explanations for the depen-dence of adhesion strength on FN7–10 clustering. Two logicalpossibilities are that 1) the proximity of the integrin cytoplasmicdomains enables the recruitment of a stabilizing protein complexwhich increases collective or individual adhesion strength or 2)fibronectin clustering increases lateral integrin interactions (35),promoting �5�1 integrin recruitment as observed (Fig. 6) andincreasing the fraction of FN7–10 molecules bound to integrins.In both cases, clustering would also reduce integrin and fibronec-tin diffusion after bond breakage, facilitating their reattachmentand enhancing adhesion. These mechanisms would not requirefurther binding of integrins to talin or the cytoskeleton to permitadhesion, and would therefore explain why talin or myosin IIdepletion did not affect adhesion strength [Fig. S2 and previouslyobserved (26)] or prevent initial cell spreading (18, 20). Fromthese measurements, it is thus clear that fibronectin fibers orother fibronectin aggregates would have much greater adhesionstrength than single molecules.

These studies provide insights into the role of talin in adhe-sions. Although adhesion strength does not depend on talin, talinis necessary for coupling to the actin rearward flow. Further,while talin is known to induce �v�3 integrin clustering (36), herewe observe that this mechanism operates in both directions. Thatis, an externally induced integrin clustering also leads to talinbinding and recruitment, and this is required to mechanicallylink the adhesion site to the cytoskeleton and to enable rein-forcement. Talin could mediate reinforcement by differentmechanisms, such as inducing further integrin recruitment underforce. However, our observation that talin mediates both rein-

forcement and cytoskeletal connection strongly suggests thattalin reinforces adhesion sites by increasing the mechanicalconnection to the cytoskeleton (and thus to the cell actomyosinmachinery) under force. Indeed, we recently observed that talinincreases its binding to the cytoskeletal protein vinculin whensubjected to force (37). Nevertheless, we found that initialspreading on fibronectin is not dependent on talin (18), and weobserve here that even though talin is involved in integrinactivation (8, 9), its role is mainly that of a scaffold enablingmechanotransduction and linkage to the cytoskeleton, and notan adhesive one. This could be explained by the recent finding(8) that talin-mediated integrin activation is more importantwith �3 integrins (which had no role in adhesion strength) thanwith �1 integrins, the major adhesion strength bearers. Even iftalin does not support adhesion per se, its enabling of reinforce-ment could increase long term adhesive strength through adhe-sion molecule recruitment and focal adhesion maturation (18,38). This could explain the rounding up of talin1�/� cells treatedwith talin2 siRNA after initial spreading (18) or the detachmentbetween integrins and actin in talin-null drosophila embryos(38). It must be noted that we cannot rule out the possibility thatremaining non-silenced talin2 in talin1�/� cells transfected withtalin2 siRNA still contributed to adhesion strength. However,the strong deficiencies of those cells in reinforcement andspreading (18) suggest that any important effect of talin inadhesion strength would have been clearly detectable. Thus, theresults that inhibition of talin [and myosin II, Fig. S6 and (26)]do not affect adhesion strength indicate that the molecular linksthrough which cells generate internal forces, responsible forreinforcement and rearward transport, are not necessary to resistexternal forces. This function would be provided by othermechanisms, which we show to include �5�1 integrins.

We suggest a model for the mechanics of fibronectin-cellcontacts in which fibronectin clustering leads to integrin binding,clustering and recruitment. Force applied to the cell is thensupported mainly by clustered �5�1 integrins, which can resistforces because broken integrin-fibronectin links quickly rebinddue to reduced diffusion of neighboring binding sites. Fibronec-tin-�v�3 links are however less stable, and their more frequentforce-induced binding/unbinding events might initiate signaltransduction, leading to adhesion reinforcement. Finally, bindingof talin to integrin clusters mechanically connects the adhesionsite to the cytoskeleton and enables reinforcement. Thus, whileadhesion strength is mediated mainly by integrin �5�1, reinforce-ment and mechanotransduction require �v�3, and talin mechan-ically connects the adhesion site to the cytoskeleton.

Materials and MethodsCell Culture. The MEF and derived Myosin IIA knock-down fibroblast cell lines[previously described (20) as RPTP��/�] were cultured in DMEM supplementedwith 10% fetal bovine serum (FBS). The MEF talin1�/� fibroblast cell line[previously described (18)] was cultured in DMEM/F12 medium supplementedwith 15% FBS (all from Gibco).

Bead Preparation. Carboxylated magnetic beads of 2.8-�m diameter (Invitro-gen) were first avidinated by incubating for 2 h 15� at 0.8% (wt/vol) in a sodiumacetate buffer (0.01 M, pH 5.0) with 1.25 mg/mL neutravidin (Invitrogen) and2.5 mg/mL carbodiimide (Polysciences) at room temperature. Beads were thenwashed in phosphate buffer saline (PBS), incubated with ethanolamide solu-tion for 30�, washed with PBS again, and resuspended in nonbiotinylated BSAsolution (10 mg/mL) in PBS. Avidinated beads were then incubated with 50�g/mL of biotinylated FN7–10 (in monomeric or pentameric form) and bio-tinylated BSA (Sigma) for 3 h (at 500 �g/mL unless otherwise specified),washed with PBS, and incubated overnight at 4 °C with 10 mg/mL biotinylatedBSA to passivate remaining unbound neutravidin in beads. Beads were thenwashed in 10 mg/mL BSA in PBS and stored at 4 °C until use. Beads were incontinuous rotation in all steps to prevent settling.

Quantification of Bead Functionalization. To quantify the amount of FN7–10 inbeads, beads were prepared as for measurements, mixed with 1� Laemli

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buffer (Bio-Rad), boiled at 100 °C for 10 min and loaded onto 7.5% polyacryl-amide gels (Bio-Rad). A gradient of known concentrations of FN7–10 wasprepared and loaded in the same way. Protein was then transferred to anOptitran reinforced nitrocellulose membrane (Whatman), which was blockedwith 5% dry milk-Tris buffered saline (TBS) and incubated with mouse anti-fibronectin binding domain antibody (Chemicon) overnight at 4 °C. Afterincubating for 1 h at room temperature with anti-mouse horseradish perox-idase (Jackson Laboratories), the membrane was blocked with TBS with 0.05%Tween-20 (Fisher Biotech) for 1 h. Marked protein was detected with ECLWestern blotting detecting reagents (Amersham Biosciences) on Kodak Bi-oMax XAR film. The signal was quantified using Imagej software, and wasconverted to protein concentrations using the reference FN7–10 gradient gellanes.

Force Measurements. To apply forces to magnetic beads, a previously de-scribed magnetic tweezers apparatus (12) was used. Briefly, an electromagnetwith a sharpened ferromagnetic core was used to apply a strong magneticfield gradient, generating a force on the beads. The force exerted by thetweezers was calibrated from the velocity of beads in liquids of knownviscosity measured as a function of the tip-bead distance and applied current.For force measurements, fibronectin-coated beads were deposited on cover-

slips silanized with 1,1,1,3,3,3,-hexamethyldisilazane (Aldrich) and coatedwith 40 �g/mL laminin (Sigma) for 2 h at 37 °C. Coating with laminin (withintegrin receptors different to fibronectin) prevented any effect of �5�1 or�v�3 inhibition on cell spreading. In Fig. S3, coating was done with 10 �g/mLvitronectin (Invitrogen). Cells were then trypsinized, resuspended in Ringerbuffer solution (150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 20 mMHEPES, and 2 g/L glucose, pH 7.4) for 30 min at 37 °C for recovery, and platedon the coverslips. The system was then mounted on a motorized 37 °C stageon an Olympus IX81 fluorescence microscope. DIC images and videos weretaken with a 60� objective and a Cascade II CCD camera (Photometrics) at afrequency of 12.8 Hz.

Constructs, transfection, antibodies, and chemicals used, and analysis ofbead recruitment, bead stiffness and detachment data, and statistics areavailable as SI Methods.

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 themembers of the Sheetz laboratory. This work was funded by National Insti-tutes of Health Roadmap for Medical Research Grant PN2 EY016586 (toM.P.S.); Marie Curie International Outgoing Fellowship within the 7th Euro-pean Community Framework Program Grant PIOF-GA-2008-219401 (toP.R.C.). and a National Institutes of Health award (to A.d.R.).

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