SUMMARY

Integrins are heterodimers (e.g., a5b1) that mediate cell adhesion in many physiological processes. Binding of integrins to ligands provides anchorage and signals for the cell. However, how force regulates integrin/ligand dissociation is unclear. Atomic force microscopy was used to measure the force dependence of lifetimes of single bonds between FN and integrin a5b1. First, lifetime-force relationships demonstrated that force prolonged bond lifetimes in the 10-30 pN range, a behavior called catch bonds. Changing divalent cations from Ca2+/Mg2+ to Mg2+/EGTA and to Mn2+ caused more pronounced catch bonds. A truncated a5b1 construct containing the headpiece but not the legs (tra5b1-Fc) formed much longer-lived catch bonds that were not affected by cation changes. Bindings of two activating mAbs, 12G10 and TS2/16, leftshifted the catch bond and converted catch bonds to slip bonds, respectively. Second, FNIII7-10/a5b1-Fc/GG-7 bond was stretched to 30 pN and then relaxed to 7 pN at which the bond’s lifetime was measured. The strong bond state induced by the 30 pN stretching stayed stable even after the force was reduced to 7 pN. In other words, lower the force would not weaken FNIII7-10/a5b1-Fc bond once it had been stretched. Similar behaviors were observed for FNIII7-10/tra5b1-Fc and FNIII7-10/ma5b1 interactions. In addition, the efficiency of the force to induce such a strong bond state for FNIII7-10/a5b1-Fc interaction in 2 mM Mg2+/EGTA condition was characterized. The probability of force to induce the strong bond state increased as force increased and when the force reached 26 pN, all bonds were transit to the strong state. Moreover, reversible unbending of a5b1 binding with FNIII7-10 under mechanical force were observed, which proved that integrin bending and unbending was dynamic. Importantly, integrin could restore bent conformation even when engaged with its ligand, providing a mechanism for mechanotransduction. Third, structural changes of a5b1 under force were observed. The structural changes did not change the trend of lifetime-force relationships of FNIII7-10/a5b1/GG-7 bond. Moreover, the lifetime for the structural changes to occur and molecular length changes caused by them were characterized.