integrins are heterodimeric membrane receptors that mediate cell adhesion to ecm or to another cell ( hynes , 2002 ) . ligand binding provides transmembrane mechanical links to transmit forces from extracellular contacts to intracellular structures ( e.g. , the cytoskeleton ) and signals for a wide variety of cellular processes . for example , binding of integrin 51 to fibronectin ( fn ) plays an important role in fibroblast spreading and motility ( akiyama et al . , 1989 ) , t cell migration ( shimizu et al . , 1990 ) , osteoblastic and myogenic proliferation , and differentiation ( garcia et al . , 1999 ) . integrins are often expressed on cells in an inactive , low affinity state with slow on rate and/or fast off rate for ligand binding , but they can be activated to high affinity states with fast on rates and/or slow off rates ( hynes , 2002 ; luo et al . , 2007 ) . activation can also be triggered by divalent cations and by binding of activating mabs to cell surface or purified integrins ( humphries , 2000 ) . affinity regulation in integrins is thought to be allosteric ( hynes , 2002 ; luo et al . , 2007 ) . the overall shape of the integrin molecule is that of a large head region ( the headpiece ) supported on two long legs . in the low affinity state , integrin legs were observed to have bent knees , which were straightened upon activation ( xiong et al . , 2001 ; takagi et al . , therefore , it seems reasonable to speculate that tensile force applied via a bound ligand may induce unbending of the knees , thereby converting the integrin from a low affinity state with short bond lifetimes to a high affinity state with long bond lifetimes ( chigaev et al . , 2003 ; zhu et al . , 2005 ; alon and dustin , 2007 ; luo et al . , 2007 ; mcever and zhu , 2007 ) . indeed , recent studies have provided experimental support for force - enhanced integrin function ( astrof et al . , 2006 ; woolf et al . , 2007 ; friedland et al . , 2009 ) . also , steered molecular dynamics simulations have suggested how force activation of integrin might occur ( jin et al . , 2004 ; puklin - faucher et al . , 2006 ) . the counterintuitive behavior in which force prolongs bond lifetimes is called catch bonds , which is in contrast to the ordinary slip bond behavior where force shortens bond lifetimes ( dembo et al . , 1988 ) . catch bonds have been demonstrated in interactions between selectins and ligands ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) , actin and myosin ( guo and guilford , 2006 ) , fimh receptor and mannose ( yakovenko et al . , 2008 ) , and glycoprotein ib ( gpib ) and von willebrand factor ( vwf ; however , loading rate - dependent rupture force measurements with atomic force microscopy ( afm ) force - ramp experiments and dynamic force spectroscopy analysis have not revealed catch bonds for integrin  ligand interactions ( li et al . , 2004 ) . using afm force - clamp experiments , we measured lifetimes of single fn51 bonds at forces as low as 4 pn . catch bonds were observed in < 30 pn . changing divalent cations altered lifetimes in the same force range and binding of a mab that reports the extended conformation of 51 , but the catch bonds remained . truncating the 51 leg regions further prolonged bond lifetimes and abolished the response of the lifetime versus force curve to divalent cations ; nevertheless , catch bonds were still observed , showing that leg extension is not required for catch bonds . two activating mabs that bind the headpiece shift catch bonds to a lower force range , indicating that force - induced activation of the headpiece is involved in catch bond formation . thus , catch bond formation appears to involve force - assisted activation of the headpiece but not integrin extension . using afm ( fig . 1 a ) , we measured interactions between an fn fragment ( fniii710 [ fn fragment encompassing the 710th type iii repeats ] ) and a recombinant 51 consisting of either the full extracellular portion ( 51-fc ) or only the headpiece ( truncated 51 [ tr51]-fc ) fused with fc ( coe et al . , 2001 ; we have shown previously that the recombinant proteins closely mimic the function and conformational regulation of the native integrin ( coe et al . , 2001 ; mould et al . , 2002 , 2003a , b , 2005 ) . to avoid uncontrolled adsorption of integrin via different domains , which could produce variable results and restrict conformational changes , tr51-fc was captured by fab of an anti - fc mab ( gg-7 ) preadsorbed on a polystyrene petri dish ( fig . the petri dish was driven by a piezoelectric translator ( pzt ) to contact fniii710 adsorbed on a cantilever tip ( fig . 1 d ) of adhesion was detected upon retraction of the pzt , which was then clamped at a desired force for lifetime measurement ( fig . a laser is focused on the back of cantilever end and bounced onto a photodiode to measure force on the tip that bends the cantilever . a petri dish is mounted on a pzt with an integrated capacitive sensor to allow for distance control with subnanometer precision . molecules depicted represent a composite of several adsorbed or captured on the cantilever tip or the petri dish . extended and bent 51-fc , depicted as heterodimers of an  ( light blue ) and a  ( red ) subunit fused to an fc ( yellow ) , and tr51-fc , consisting of the  propeller ( teal ) and thigh ( dark green ) domains of the  subunit as well as the a ( pink ) , hybrid ( dark blue ) , and plexin / semaphorin / integrin ( tan ) domains of the  subunit fused to an fc , were captured by an anti - fc mab ( gg-7 ) fab ( blue ) preadsorbed on the petri dish . in some experiments , these were replaced by an anti - fn mab ( hfn7.1 ; brown ) captured by an anti  mouse fc antibody ( orange ) preadsorbed on the petri dish . fniii710 ( purple ) was adsorbed on the cantilever tip . in some control experiments , the petri dish was moved up by the pzt to contact the cantilever tip ( blue trace ) , immediately retracted to a small distance ( green trace ) from the tip to reduce nonspecific adhesion , held at this distance for 0.5 s to allow for bond formation ( brown trace ) , and retracted away from the tip to detect adhesion ( red trace ) . the trace illustrates a contact cycle without binding where the retraction curve returned to zero force upon petri dish retraction . the color codes are the same as those in c , which illustrates a contact cycle with binding and lifetime measurement . once the pulling force reached a preset value ( indicated ) , a feedback loop was triggered to keep the cantilever deflection at the set point . the lifetime at that force ( indicated ) was measured until bond failure , signified by the springing back of the cantilever to the level of zero mean force . the bending configurations of the cantilever are depicted with colors matching the corresponding colors of the traces in c and d. experiments were performed in 1 mm ca plus 1 mm mg ( ca / mg ) , 2 mm mg plus 2 mm egta ( mg / egta ) , or 2 mm mn in the absence or presence of blocking or activating mabs or a competing peptide . at the same gg-7coating concentrations , binding of 51-fc ( fig . 2 c ) to fniii710 became progressively more frequent when the cation condition was changed from ca / mg to mg / egta and to mn . this was corroborated by flow cytometry data showing that mg / egta and mn increased the staining of 51-fc  coated beads by a mab that reports unbending of the knees ( 9eg7 ; fig . binding was specific , as it was abolished by addition to the solution of mab hfn7.1 , which blocked the integrin - binding site in fniii710 or an rgd - containing peptide cyclo(-grgdsp ) , which competed with the rgd loop in fniii710 for 51 binding ( fig . ( < 20% ) , a requirement for most adhesion events to be mediated by single bonds , progressively lower gg-7coating concentrations were used for experiments in ca / mg , mg / egta , and mn to compensate for the progressively higher binding affinities of tr51-fc ( fig . binding was abrogated when the cantilever tips coated with fniii710 were switched to those coated with bsa or when the petri dishes functionalized with integrins were switched to those that were not functionalized ( fig . frequencies of adhesion between 10 g / ml fniii710 adsorbed on cantilever tips and 10 g / ml 51-fc ( a ) or tr51-fc ( c ) captured by 15 or 100 g / ml gg-7 fab precoated on petri dishes in buffer containing the indicated cations without or with cyclo(-grgdsp ) or hfn7.1 in solution were enumerated in 100 tests for each spot and presented as mean  sem of 35 spots . frequencies ( measured with the same protocol as in a and c ) of adhesion between 10 g / ml fniii710 coated on cantilever tips and 10 g / ml 51-fc ( b ) or tr51-fc ( d ) captured by gg-7 fab preadsorbed on petri dishes with indicated concentrations ( closed bars ) are compared with those between bsa - coated tips and tr51-fc  functionalized petri dishes ( open bars ) and with those between fniii710-coated tips and gg-7coated petri dishes without incubating with tr51-fc ( hatched bars ) in the indicated cation conditions . mechanical regulation of fn51 dissociation was quantified by the force dependence of mean lifetimes of mostly single fniii71051 bonds measured in each of the three cation conditions ( fig . 3 , a  c , circles ) . as force increased , lifetime first decreased to a minimum , then increased to a maximum , and decreased again , exhibiting a triphasic transition from slip bonds to catch bonds and then to slip bonds again . the first slip bond regimen was most clearly observed in ca / mg ( fig . the much less frequent nonspecific binding between bsa - coated cantilevers and polystyrene petri dishes functionalized with 51-fc contributed negligibly to these lifetime versus force curves , as most of these events were ruptured at forces < 20 pn ( fig . s2 a ) , and those that survived the ramping had very short lifetimes ( fig . ( a  c ) plots of lifetime versus force of 51-fc  functionalized petri dish dissociating from fniii710-coated cantilever tips ( circles ) in ca / mg ( a ) , mg / egta ( b ) , and mn ( c ) . ( d ) a qualitatively similar plot ( triangles ) of m51 reconstituted into lipid bilayer dissociating from fniii710-coated cantilever tips in mg / egta confirmed the catch bond observation . also plotted in a c is the lifetime versus force curve of fc dissociating from gg-7 ( squares ) . for b and c , the black and gray curves overlap at forces > 30 pn , indicating that measured lifetimes beyond 30 pn were caused by fc also shown in c is a lifetime versus force plot ( diamonds ) of 51-fc  functionalized petri dish dissociating from bsa - coated cantilever tips measured in mn . ( e ) schematic of the molecular arrangement indicating possible dissociation loci between fniii710 and 51-fc or between 51-fc and gg-7 . ( f ) schematic of the molecular arrangement for experiments that measured the capturing strength of the fc error bars indicate mean  sem . to control for potential artifacts of the chimeric integrin that fuses the extracellular portions of the 5 and 1 chains with a human igg fc region ( coe et al . , 2001 s2 a ) of fniii710 to membrane 51 ( m51 ) purified from human smooth muscle cells reconstituted in glass - supported lipid bilayers in mg / egta . although the lifetimes were shorter , the force - dependent curve of m51 exhibited the same triphasic pattern ( fig . thus , catch bonds were observed at forces ranging from 1030 pn for fn interacting with 51-fc and m51 . because 51-fc was captured by preadsorbed gg-7 , rupture of the molecular complex might result from dissociation of the fn51 or fc  gg-7 bond ( fig . we neglect potential detachment of fniii710 from the cantilever tip or of gg-7 from the petri dish because physioadsorption of proteins is generally much stronger than specific protein  protein interactions ( rief et al . , 1997 ) . to determine the rupture loci , we overlaid the force - dependent lifetimes of directly adsorbed 51-fc interacting with gg-7 ( fig . 3 f ) on each panel of fig . 3 ( a  c , squares ) . slip bonds were observed over the entire force range tested . the mean lifetimes at forces < 25 pn were much longer than those of fniii710 interacting with captured 51-fc , indicating that the observed catch bonds were characteristic of the fn51 bond rather than the fc however , at forces > 30 pn , the black circle curves in fig . 3 ( b and c ) became indistinguishable from the gray square curve , suggesting that the second slip bonds of the former curves result from dissociation of the fc gg-7 bond rather than the fn51 bond . because the dissociation rate of two bonds in series equals to the sum of the two individual dissociation rates , the lifetimes of these two bonds in series would have been significantly shorter than either bond measured separately if lifetime of the fn51-fc bond were comparable with the fc the fact that the lifetime versus force curves of the fn51-fc  gg-7 and 51-fc gg-7 bonds were indistinguishable shows that the fn51 bond lifetimes in mg / egta or mn were substantially longer than the fc they might continue to behave as catch bonds at forces > 30 pn before ( and if ) it transitioned to slip bonds . however , the real differences of fn51 bond lifetimes between ca / mg and mg / egta or mn conditions may be much greater than those apparent in fig . 3 ( a  c ) . for comparison , we also measured force - dependent lifetimes of 51 and fniii710 bound to their respective blocking mabs , p1d6 and hfn7.1 ( fig . 4 , schematics ) , which were also specific to the antigen  antibody interactions ( fig . s2 , c and d ) . similar to the fc  gg-7 interaction and consistent with other antibody  antigen interactions characterized previously ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) , slip bonds were observed in both cases ( fig . 4 ) as the mean lifetimes decreased monotonically with increasing force in the same range where the triphasic transitions between slip and catch bonds were observed for 51 interacting with its physiological ligand ( fig . 4 , compare a with b ) , revealing different interaction characteristics of the different antibody lifetimes of antibody  antigen bonds . ( a and b ) plots of lifetime ( mean  sem ) versus force of interactions between 51-fc  functionalized petri dish and p1d6-coated cantilever tips ( a ) and between hfn7.1 captured by anti a large percentage of 51-fc appeared to exist in a bent conformation in ca / mg , but more of the integrin became extended in mg / egta and mn ( fig . yet in all three cation conditions , lifetimes at low forces ( < 10 pn ) were similarly short ( < 2 s ) . as force increased from 1030 pn , catch bonds were observed , but lifetimes were prolonged less by force in ca / mg ( fig . these data indicate that catch bonds may not result from a force - induced unbending of the integrin . to obtain more definitive evidence 3 ( a  c ) using tr51-fc that contains only a five - domain headpiece of the integrin fused with fc ( coe et al . , 2001 ; mould et al . , 2002 ) . as anticipated , catch bonds were still observed , confirming that the integrin legs and the unbending conformational change were not required for the fn51 catch bonds ( fig . 5 , a  c ) . interestingly , in the 1025-pn range , truncating the integrin leg regions enabled force to prolong lifetimes to a greater extent than the integrin with legs ( fig . 5 , the lifetime versus force curves in the catch bond regimen were quite similar for all three cation conditions ( compare fig . 5 , a  c ) however , similar to the fniii71051 case , the force where the fniii710tr51 catch bonds might transition to slip bonds could not be determined , for the mean lifetimes of the fniii710tr51-fc gg-7 bond at a force > 25 pn in all three cation conditions ( fig . 5 , a 3 ) , indicating that these represented lifetimes of the fc  gg-7 bond rather than the fniii710tr51-fc bond . a  c ) plots of lifetime ( mean  sem ) versus force of tr51-fc  functionalized petri dish dissociating from fniii710-coated cantilever tips ( circles ) in ca / mg ( a ) , mg / egta ( b ) , and mn ( c ) . data from fig . the lifetime versus force curves of 51-fc  functionalized petri dish dissociating from fniii710-coated cantilever tips are shown as triangles , and 51-fc  coated cantilever tips dissociating from gg-7functionalized petri dish are shown as squares . ( d ) schematic of the molecular arrangement indicating possible dissociation loci between fniii710 and tr51-fc or between tr51-fc and gg-7 . to explore the structural mechanism of the observed catch bonds , we measured fniii71051-fc bond lifetimes in mn in the presence of ts2/16 ( fig . 6 a ) or 12g10 ( fig . 6 b ) . 6 d , schematic ) to further activate integrins by shifting or stabilizing the position of the 1 helix known to be critical for integrin activation ( mould et al . both mabs left shifted the catch bond region , but ts2/16 yielded longer lifetimes at low forces ( fig . interestingly , ts2/16 also left shifted the fniii710tr51-fc catch bond region but did not prolong lifetimes as much as it did for the fniii71051-fc case ( fig . these data emphasize the importance of the a domain 1 helix and link the fniii710tr51 catch bonds to 51 activation . ( a  c ) lifetime ( mean  sem ) versus force plots ( circles ) of 51-fc  functionalized petri dish dissociating from fniii710-coated cantilever tips in mn and 10 g / ml ts2/16 ( a ) or 12g10 ( b ) or tr51-fc  functionalized petri dish dissociating from fniii710-coated cantilever tips in mn and 10 g / ml ts2/16 ( c ) . for comparison , data from fig . 3 c are replotted in a and b , and some of the data from fig . 5 c are replotted in c , where the lifetime versus force curves of tr51-fc  functionalized petri dish dissociating from fniii710-coated cantilever tips are shown as triangles , and 51-fc  coated cantilever tips dissociating from gg-7functionalized petri dish are shown as squares . ( d ) schematic of the molecular arrangement indicating binding sites for ts2/16 and 12g10 . catch bonds represent unusual , counterintuitive behaviors that have recently been observed in selectin  ligand ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) , actin  myosin ( guo and guilford , 2006 ) , fimh  mannose ( yakovenko et al . , 2008 ) , and gpibvwf ( yago et al . , 2008 ) interactions . based on the integrin structures and their conformational change models , it has been speculated that integrin  ligand interactions may also behave as catch bonds ( chigaev et al . , 2003 ; zhu et al . , 2005 ; alon and dustin , 2007 ; luo et al . , 2007 ligand catch bonds ( zhang et al . , 2002 , 2004 ; li et al . , 2003 ) . in the previous work , force - ramp experiments were used to measure ramp rate - dependent rupture forces , which were analyzed by dynamic force spectroscopy , assuming that dissociation occurs along a single pathway and off rate increases exponentially with increasing force as modeled by bell ( 1978 ) , which precluded catch bonds . using afm force - clamp experiments , we observed triphasic force - dependent bond lifetimes that deviate from the bell model , demonstrating fn51 catch bonds ( figs . 3 , 5 , and 6 ) . it is known that integrin - mediated adhesion can be strengthened by integrin clustering or binding of multiple integrins cooperatively , which can be induced by force ( galbraith et al . , however , this is unlikely the mechanism for the observed catch bonds for the following reasons . first , ( tr)51-fc molecules were captured by gg-7 fab , which was unlikely to adsorb on petri dishes as clusters . nor was the fniii710 likely to adsorb on afm tips as clusters . without preclustering , without active cellular mechanisms to cluster the molecules , and without lateral mobility , it is unlikely that integrins bind in clusters in our experiments . second , using the same afm setup in the control experiments , we observed only slip bonds for the 51-fc gg-7 interaction was monomeric ( because fab was used ; fig . 3 f ) , 3 a , schematic ) , and fniii710hfn7.1anti  mouse fc interaction might be multimeric ( because whole hfn7.1 and anti  mouse fc were used ; fig . 3 b , schematic ) . third , we examined the correlation of ( or the lack thereof ) bond lifetime with molecular stiffness ( fig . we have previously shown that the stiffness of multiple bonds is multiple times of that of a single bond ( sarangapani , 2005 ) , which predicts a positive correlation between lifetime and stiffness if the longer lifetimes were caused by greater bond multiplicity . however , such correlation was not observed ( fig . s3 , a and b ) . to the contrary , the mean stiffness values for molecular complexes that had lifetimes clustered around 0.3 , 2.8 , and 10 these data ruled out higher multiplicity of bonds as the cause for longer lifetimes at high forces than the lifetimes at low forces in the catch bond regime . finally , we have previously shown that formation of dimeric bonds can not generate catch bonds if the corresponding monomeric interaction is a slip bond . the effects of dimeric bonds are to shift the lifetime versus force curve of the monomeric bond rightward toward doubling the force and upward toward doubling the lifetime ( marshall et al . , 2003 ) . our flow cytometry data suggest that more 51-fc became extended in mg / egta and mn than in ca / mg ( fig . s1 ) , which is consistent with previous flow cytometric ( humphries , 2000 ) , crystallographic ( xiong et al . , 2001 ) , and electron microscopic ( takagi et al . , 2002 ; zhu et al . , the affinity of fn51 binding was low in ca / mg but high in mg / egta or mn ( mould et al . , 1995 ) , which was manifested as different adhesion frequencies ( fig . 2 , a and c ) . at low forces ( < 10 pn ) , fn51 bonds dissociated rapidly ( lifetimes < 2 s ) in all three cation conditions ( fig . c ) , suggesting that mg / egta or mn increased the on rate for association but did not significantly impact the off rate for dissociation . these are consistent with our previous kinetic measurements of integrin l2 interacting from intercellular adhesion molecule 1 under these cations , which showed fast force - free off rates and a much greater responsiveness to cation conditions of on rates than off rates ( zhang et al . , 2005 ) . in previous tension - free studies , mn has often been considered to be able to activate integrin to a high affinity state for ligand binding ( mould et al . , 1995 ; takagi et al . , 2002 ) , which corresponds to the bond state at low forces in our experiment . then , force is able to further strengthen the bond , inducing new bond states previously not identified ( friedland et al . , 2009 ) . it has been suggested that force applied to a bent integrin may straighten it to an extended integrin , thereby giving rise to catch bonds ( chigaev et al . 2005 ; alon and dustin , 2007 ; luo et al . , 2007 ; mcever and zhu , 2007 ) . however , catch bonds were observed not only for fn interacting with 51 ( fig . c ) in three different cation conditions but also for fn interacting with tr51 that lack the leg regions ( fig . 5 , a c ) , which indicates that force - induced unbending is not a required conformational change for fn51 catch bonds . an essential feature of integrin activation is a swing of the  subunit hybrid domain away from the  subunit ( takagi et al . , 2002 ; , it has been proposed that , without a lateral force to separate the integrin legs , pulling on an extended integrin would prevent the hybrid domain from swinging outwards , thereby stabilizing the inactive conformation of the headpiece ( zhu et al . , 2008 ) . however , in our experiments , no lateral force was applied to separate the integrin legs , which were restrained . yet , pulling on 51 via a bound fn prolonged bond lifetimes at low forces , indicating that force activates rather than deactivates 51 . it is possible that , at least in our system , the bond strengthening caused by pulling on the 1 helix connection may outweigh the effects of force on hybrid domain movement . similar to the recombinant 51-fc fusion protein , native 51 purified from cell membrane and reconstituted into glass - supported lipid bilayers also formed catch bonds with fn in the same force range , although the apparent lifetimes were shorter ( fig . for the majority of the adhesion events , the shorter lifetimes may not be caused by extraction of 51 from the lipid bilayer because the running adhesion frequency remained stable in a large number of repeated contacts ( fig . s4 b ) , even when the same cantilever tip was used to contact the same lipid bilayer location , indicating that neither the tip nor the bilayer lost functionality over time ( chesla et al . , 1998 ) . however , it is possible that some very long lifetimes ( > 10 s ) could be cut short by extrapolating of integrin from the bilayer . in addition , more 51-fc might be in an extended conformation than m51 in the same cation condition , resulting in longer lifetimes for the catch bonds of fn with 51-fc than with m51 ( mould et al . , 2005 ) . as discussed in the preceding paragraph , in the bent conformation , although catch bonds were observed for both interactions of fn with 51-fc and m51 , the causes of their lifetime differences remain unclear and require further studies . binding of ts2/16 or 12g10 left shifted the fn  tr51-fc catch bond region by prolonging lifetimes at low forces ( fig . both mabs bind at or near to the 1 helix of the a domain ( fig . 6 d ; mould et al . , 2002 ) , suggesting that this region could be important in regulating the catch bond behavior . in addition to the rgd - binding site at the a domain of the 1 subunit , the  propeller of the 5 subunit may bind the synergy site at the fniii9 domain to strengthen the fn51 interaction ( garcia et al . , 2002 ; mould et al . , 2003b ; friedland et al . , 2009 ) . it has recently been proposed that the fn51 bond can be switched from a relaxed to the tensioned states in response to mechanical force through engaging the synergy site in fn ( friedland et al . , 2009 ) . multiple sites have also been proposed for ligand binding to integrin headpieces ( hynes , 2002 ; liddington and ginsberg , 2002 ; friedland et al . thus , fn51 catch bonds could also involve force - induced binding of multiple sites that strengthen the molecular complex . interestingly , the natural log ( number of measurements with a lifetime > t ) versus t plots for the fn51-fc bond exhibited multiple line segments ( fig . c ) , which are in contrast to the more linear plots of the 51-fc . the negative slope of a line reflects the off rate of the subpopulation of dissociation events associated with that line ( marshall et al . , 2003 ) . multiple line segments suggest multiple states of single fn51-fc bonds rather than multiple fn51-fc bonds because 51-fc were captured by gg-7 fab randomly and sparsely adsorbed and because the lifetimes were measured from single rupture events . similar multiple - bond states were observed for fimh  mannose catch bonds ( thomas et al . , 2006 ) . these are different from the selectin  ligand catch bonds that show single lines in such plots ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) instead of the aforementioned allosteric , multisite , and multistate models , a sliding rebinding model was proposed for selectin  ligand ( lou et al . , 2006 ; lou and zhu , 2007 ) and gpibvwf ( yago et al . , 2008 ) these mechanisms are not mutually exclusive and may work together to give rise to integrin  ligand catch bonds . they also transduce signals bidirectionally from inside out and from outside in across the cell membrane . such a dual role makes integrins prime candidates for force - sensing molecules in mechanotransduction ( schwartz and desimone , 2008 ) . catch bonds provide a physical mechanism for force sensing if different bond lifetimes correspond to different activation states that transduce distinct signals . the ability of integrin  ligand bonds to be strengthened by force may be of great importance not only for leukocyte trafficking , which occurs under hydrodynamic forces , but also for the migration of many other cell types , which involves cyclic adhesion and detachment between the cell and the ecm . catch bonds may provide a mechanical mechanism for the cell to regulate adhesion by applying different forces at different times and locations when and where different adhesion strengths are desired . recombinant 51-fc and tr51-fc chimeras were generated by chol761h cells that were transiently transfected with cdna constructs encoding the human 5 ( fitzgerald et al . , 1987 ) and 1 ( coe et al . , 2001 ) subunits or tr5 and tr1 subunits ( coe et al . , 2001 ) . fniii710 with a biotin tag at the n terminus was produced using standard recombinant dna techniques ( petrie et al . , 2006 ) . anti-51blocking ( p1d6 ) and activating ( ts2/16 ) mabs were obtained from millipore , reporting mab ( 9eg7 ) was obtained from bd , and another activating mab ( 12g10 ) was obtained from abcam . the anti - fn mab ( hfn7.1 ) was obtained from developmental studies hybridoma bank . the anti  human fc - capturing mab ( gg-7 ) was obtained from sigma - aldrich . the afm is a modification of a previously described system ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) it consists of a pzt on which a petri dish is directly mounted ( fig . 1 a ) . the pzt has a capacitive feedback control that gives subnanometer position resolution . a laser ( oz optics ) focused on the end of the cantilever ( tm microscopes ) is deflected onto a photodiode ( hamamatsu photonics ) to measure cantilever deflection , which is converted to force using the cantilever spring constant . spring constant ( 312 pn / nm ) for each cantilever was calibrated in situ using thermal fluctuation analysis ( hutter and bechhoefer , 1993 ) . a personal computer with a data acquisition board ( national instruments ) was used to control movements of the pzt and to collect signals from the photodiode . labview ( national instruments ) was used as the interface between the user and the data acquisition board . to measure the interaction of 51-fc or tr51-fc with fniii710 ( fig 4 a , schematic ) , cantilever tips were incubated with 1020 g / ml fniii710 or p1d6 overnight at 4c ( fig . 1 b ) . after rinsing with tbs ( 25 mm tris - hcl and 150 mm nacl , ph 7.4 ) , the cantilevers were incubated for 15 min at room temperature in tbs containing 1% bsa to block nonspecific adhesion . gg-7 was cleaved into fab and fc fragments by using the fab preparation kit following the manufacturer 's instructions ( thermo fisher scientific ) . 2 ) was adsorbed on a small spot on a petri dish by overnight incubation at 4c . to capture tr51-fc , the gg-7precoated petri dish was rinsed three times with tbs and incubated with 10 g / ml tr51-fc at the desired cation condition ( ca / mg , mg / egta , or mn ) for 30 min . the petri dish was again rinsed three times with tbs and filled with 5 ml tbs plus 1% bsa and the indicated cations . in some experiments , 10 g / ml hfn7.1 , 1 mg / ml cyclo(-grgdsp ) ( anaspec , inc . ) , or 10 g / ml ts2/16 or 12g10 ( fig . 6 d ) were added to the buffer . to measure the interaction of fniii710 with hfn7.1 ( fig . 4 b , schematic ) or 51-fc with gg-7 fab ( fig . 3 f ) , fniii710 or 51-fc was adsorbed on cantilevers and treated as described in the previous paragraph . 10 g / ml goat anti mouse fc polyclonal antibody or 20 g / ml fragmented gg-7 was adsorbed on a labeled spot on a petri dish overnight at 4c . to capture hfn7.1 , the petri dish was rinsed three times with tbs and incubated with 10 g / ml hfn7.1 for 30 min . the petri dish was again rinsed three times with tbs and filled with 5 ml tbs plus 1% bsa and the indicated cations . to measure the interaction of fniii710 with m51 , cantilevers were coated with streptavidin at 50 g / ml overnight at 4c and further functionalized by incubation with 10 l of 1 g / ml fniii710 for 15 min at room temperature . ( 1993 ) . in brief , vesicles were formed by hydrating a dried lipid film of 1,2-dimyristoyl - sn - glycero-3-phosphocholine and 1,2-dimyristoyl - sn - glycero-3-[phospho - rac-(1-glycerol ) ] ( avanti polar lipids , inc . ) in a 50:50 ratio with 0.1% triton x-100 ( thermo fisher scientific ) in tbs and mixed with m51 , resulting in a final concentration of 0.27 mg / ml ( 0.4 mm ) of lipid and 0.1 mg / ml of integrin . triton x-100 was removed by adsorption to biobeads sm-2 ( bio - rad laboratories , inc . ) at 37c for 4 h. the resulting lipid vesicle solution was stored under argon at 4c and used within several months . coverslips of 40 mm in diameter ( bioptechs ) were cleaned with a mixture of 70% 12 n sulfuric acid and 30% hydrogen peroxide by volume at 100c for 45 min , rinsed extensively with deionized water , and dried completely under an argon stream . the cleaned coverslip , which was used immediately , was placed in a petri dish , and a 4-l drop of m51-incorporated lipid vesicle solution was placed on the coverslip surface . after 20 min of incubation under a damp paper towel , the petri dish was filled with 5 ml tbs with 2 mm mg , 2 mm egta , and 1% bsa . the m51 bilayers that formed had low molecular densities to ensure their infrequent binding to the fniii710-coated cantilever tips , as required for measuring single bonds . the afm force - clamp experiments were performed by repeatedly bringing the petri dish in contact with the cantilever tip , then immediately retracting a small distance ( 05 nm ) , holding at that distance for 0.5 s to allow bond formation , and retracting again at a speed of 200 nm / s . by preventing the cantilever tip from compressing the petri dish during the time for molecular association , the nonspecific binding was greatly suppressed ( fig . 2 ; and fig . s2 , b the presence of adhesion was detected from the force - scan curves ( fig . 1 , c and d ) . a feedback system was used to clamp the force at a desired level to enable measurement of bond lifetime at constant force . approximately 50 lifetimes were measured in the full - force range ( 570 pn ) using a cantilever and a petri dish in each experiment . gg-7 interaction in 1 mm ca / mg condition , 13 experiments were performed to acquire 846 lifetimes at forces ranging from 570 pn . these were segregated into 12 force bins , each of which spanned 56 pn and contained at least 50 lifetimes . plots of natural log ( number of events with a lifetime > t ) versus t were exemplified for the fn51-fc 125 g ( 50 l ) streptavidin - coated magnetic beads ( thermo fisher scientific ) were incubated with gg-7 biotinylated using biotintag micro biotinylation kit ( sigma - aldrich ) in 1 ml pbs at a concentration of 5 g / ml for 30 min . 40 l beads were washed three times in tbs , incubated with 20 g / ml 51-fc in 200 ml tbs with desired cations for 30 min , again washed three times in tbs with appropriate cations , and incubated with 9eg7 at 10 g / ml for 30 min . 9eg7 was preconjugated with allophycocyanin using the lightning - link apc - xl conjugation kit following the manufacturer 's instructions ( innova bioscience ) . after washing three times with tbs containing the appropriate cations , s1 shows flow cytometry analysis of the expression of 9eg7 epitope by 51-fc in different cation conditions . fig .