| Integrins are heterodimers (e.g., alpha5beta 1) that mediate cell adhesion in many physiological processes. Binding of integrins to ligands (e.g., fibronectin, FN) provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. 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 a FN fragment (FNIII7-10) on the cantilever tip and an integrin alpha5beta1-Fc fusion protein captured by an anti-Fc monoclonal antibody (mAb) on a Petri dish or membrane alpha 5beta1 reconstituted in a lipid bilayer.;First, lifetime-force relationships demonstrated that force prolonged bond lifetimes in the 10-30 pN range, a counterintuitive behavior called catch bonds. Changing divalent cations from Ca2+/Mg2+ to Mg2+/EGTA and to Mn2+ caused more pronounced catch bonds by further prolonging lifetimes. In addition, flow cytometric analysis showed that Mg2+/EGTA and Mn2+ favored extended conformation of alpha5beta1 and Mn 2+ promoted hybrid domain swing-out. Therefore, extension and hybrid domain swing out did not change the catch bond behavior. A truncated alpha 5beta1 construct containing the headpiece but not the legs (tralpha5beta1-Fc) formed much longer-lived catch bonds in the same force range that were not affected by cation changes. Bindings of two activating mAbs, 12G10 and TS2/16, left shift the catch bond and converted catch bonds to slip bonds, respectively. Both mAbs, 12G10 and TS2/16, bind to alpha2 helix of A (I-like) domain in the beta1 subunit, which is close to the alpha1 helix that directed connects to the binding pocket. This suggests that the catch bonds may result from force stabilization of an active conformation of the headpiece that involves position changing of alpha1 helix. Catch bonds may provide a mechanical mechanism for the cell to regulate adhesion by applying different forces.;Second, FNIII7-10/alpha5beta1-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/alpha 5beta1-Fc bond once it had been stretched. Similar behaviors were observed for FNIII7-10/tralpha5beta1-Fc and FNIII7-10/malpha5beta1 interactions. In addition, the efficiency of the force to induce such a strong bond state for FNIII7-10/alpha5beta1-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 alpha5beta1 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 alpha5beta1 under force were observed. The structural changes did not change the trend of lifetime-force relationships of FNIII7-10/alpha5beta1/GG-7 bond. Moreover, the lifetime for the structural changes to occur and molecular length changes caused by them were characterized. |
