Cyclic Mechanical Reinforcement of Integrin–Ligand Interactions Fang Kong, Zhenhai Li, William M. Parks, David W. Dumbauld, Andrés J. García, A. Paul Mould, Martin J. Humphries, Cheng Zhu  Molecular Cell  Volume 49, Issue 6, Pages 1060-1068 (March 2013) DOI: 10.1016/j.molcel.2013.01.015 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 AFM Experiments (A) AFM schematic. (B) AFM functionalization. A composite of all molecules used is depicted. (C) Force-scan trace without adhesion. (D) A FN–α5β1-Fc–GG-7 serial bond was loaded to 5 pN and lifetime measured at that force. (E) A FN–α5β1-Fc–GG-7 serial bond was first loaded to 25 pN, then unloaded to 5 pN, and held at 5 pN for lifetime measurement. Molecular Cell 2013 49, 1060-1068DOI: (10.1016/j.molcel.2013.01.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Cyclic Mechanical Reinforcement Prolongs FN–α5β1 Bond Lifetimes (A) Mean ± SEM of indicated numbers (atop each bar) of FN–α5β1-Fc–GG-7, P1D6–α5β1-Fc–GG-7, FN–HFN7.1, and α5β1-Fc–GG-7 bond lifetimes measured at ∼5 pN without (closed bars) and with (open bars) cyclic mechanical reinforcement with an ∼25 pN peak force in the indicated conditions. (B) Schematic of a FN–α5β1-Fc–GG-7 serial bond indicating possible dissociation loci between FN and α5β1 or between Fc and GG-7. (C) Schematic of a possible molecular arrangement for experiments that measured the lifetimes of the α5β1-Fc/GG-7 capture bond. See also Figure S1. Molecular Cell 2013 49, 1060-1068DOI: (10.1016/j.molcel.2013.01.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Cyclic Mechanical Reinforcement of Cell Surface Integrins (A) Experimental setup of BFP. (B) Representative force-time traces: (i) (1) Approaching phase before contact (with zero force), (2) contact phase with a compressive force, (3) retracting phase without adhesion (the force returned to zero). (ii) Similar to (i) except that adhesion was detected in phase (3) by a tensile force. The PZT retraction stopped to hold the force constant for lifetime measurement. (iii) Similar to (ii) except that lifetime measurement was taken following a force cycle of loading to a high force then unloading to the low force. After a 10 s holding phase for lifetime measurement, the PZT retracted again to rupture the bond because force drift was often observed during measurements of exceedingly long lifetimes. (C) (Left) Adhesion frequency between FN-coated beads and α5β1-expressing K562 cells in 2 mM Mg2+/EGTA was reduced by >2-fold by 10 μg/ml of anti-FN blocking mAb (HFN7.1) and by not coating FN on the beads, indicating adhesion specificity. (Right) Adhesion frequency between ICAM-1-coated beads and αLβ2-expressing Jurkat cells in 2 mM Mg2+/EGTA was reduced by >4-fold by not coating ICAM-1 on the beads, indicating adhesion specificity. Data are presented as mean ± SEM of ≥5 cell-bead pairs each contacting 100 times. (D) Average lifetimes derived from using the two-state model to fit the lifetime distributions of FN–α5β1 (left) or ICAM1-αLβ2 (right) bonds measured at 5 pN with or without cyclic mechanical reinforcement (20 pN peak force). Data are presented as fitted mean ± SEM. Molecular Cell 2013 49, 1060-1068DOI: (10.1016/j.molcel.2013.01.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Effects of Peak Force of Single-Cycled Mechanical Reinforcement on FN–α5β1 Bond Lifetimes (A) Representative force-time traces. (B) Distributions of FNIII7-10–α5β1-Fc–GG-7 bond lifetimes at ∼5 pN with indicated peak forces (points) and their theoretical fits by the two-state model (curves). The natural log of the number of events with a lifetime ≥t normalized by the natural log of the total number of events is plotted versus t. Data for different peak forces (indicated) are separated for the sake of clarity. (C) Mean ± SEM of FNIII7-10–α5β1-Fc–GG-7 serial bond lifetimes (black squares) increased with increasing peak force before being limited by the Fc–GG-7 capture bond lifetime at >22 pN forces. Also shown are mean ± SEM lifetimes of FNIII8,10–α5β1-Fc–GG-7 bonds (purple circles) and FNIII7-10–α5β1-Fc–GG-7 bonds in the presence of 10 μg/ml HUTS-4 (orange triangles) or SG/19 (pink diamonds) versus peak forces. (D) Effects of FN synergy domain deletion and integrin hybrid domain constraining antibodies on the cell surface α5β1 (left, open bars) are comparable to those on the α5β1-Fc chimera (right, closed bars). See also Figures S2 and S4. Data are presented as mean ± SEM for α5β1-Fc (right figure) and fitted mean ± SEM for cell surface α5β1 (left figure). Molecular Cell 2013 49, 1060-1068DOI: (10.1016/j.molcel.2013.01.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Effects of Repetitive Force Cycles on FN–α5β1 Bond Lifetimes (A) Representative force-time traces showing lifetime measurements at 10 pN with 0.5, 1.5, 2.5, and 3.5 cycles of loading-unloading with a 10 pN peak force. Complete unloading to zero force was used. (B) Lifetime distributions of the measurements shown in (A). Data for different numbers of cycles (indicated) are separated for the sake of clarity. (C) Mean ± SEM lifetimes of FNIII7-10–α5β1-Fc–GG-7 (black squares) and FNIII8,10–α5β1-Fc–GG-7 (purple circles) bonds and of FNIII7-10–α5β1-Fc–GG-7 bonds in the presence of HUTS-4 (orange triangles) and SG/19 (pink diamonds) versus number of repetitive loading-unloading cycles. Molecular Cell 2013 49, 1060-1068DOI: (10.1016/j.molcel.2013.01.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Cyclic Mechanical Reinforcement of Integrin α5β1 Covalently Linked to Surface (A) Schematic of covalent tethering of α5β1-Fc to mixed SAMs of alkanthiols using standard NHS/EDC chemistry. Compared to capturing α5β1-Fc by an anti-Fc (GG-7), covalently attaching α5β1-Fc to the substrate via chemical crosslinking prevented the integrin from dissociating from the substrate. (B) Binding specificity. Data are presented as mean ± SEM. ND, not detected. (C) Distributions of FN–α5β1 bond lifetimes measured at 5 pN without (black) or with (magenta) a cyclic force of 20 pN peak (points) and their model fits (curves). (D) Distributions of FN–α5β1 bond lifetimes measured at 10 pN with 0.5 (black), 1.5 (red), 2.5 (green), and 3.5 (blue) force cycles (points) and their model fits (curves). (E) Lifetimes of the longest-lived state deduced from fitting the lifetime distributions in (C) (pink) and (D) (matched colors) with a three-state model. Molecular Cell 2013 49, 1060-1068DOI: (10.1016/j.molcel.2013.01.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 Model of Cyclic Mechanical Reinforcement (A) Reinforcing FN–α5β1 bond by a single-cycled force. (Left) Liganded integrin in the short-lived state. Key α5β1 domains are indicated along with the RGD and synergy binding sites on the FNIII 10 and 9 domains, respectively. (Middle) Force applied via the ligand primes the integrin for conformational change in the headpiece. Higher peak force (top) better primes the integrin than lower peak force (bottom). (Right) During unloading, the FN–α5β1 bond may be switched to the long-lived state (top) or may return to the short-lived state (bottom). A bond previously primed by a higher peak force is more likely to be switched to the long-lived state than a bond primed by a lower peak force, as indicated by the sizes of the arrows. (B) Reinforcing FN–α5β1 bond by multicycled small forces. (Far left) FN bound α5β1 in the short-lived state as that in (A), left. (Midleft) A loading-unloading cycle has a certain probability to switch the FN–α5β1 bond to the long-lived state (top) but also a probability not to change the short-lived state (bottom). (Midright) After another loading-unloading cycle, the bond that is already in the long-lived state stays in the long-lived state (top). However, the bond that remains in the short-lived state has a certain probability to switch to the long-lived state (bottom). (Far right) As the number of cycles increases, the FN–α5β1 bond is more and more likely to be switched to the long-lived state (top) and less and less likely to remain in the short-lived state (bottom), resulting in an accumulating effect. Molecular Cell 2013 49, 1060-1068DOI: (10.1016/j.molcel.2013.01.015) Copyright © 2013 Elsevier Inc. Terms and Conditions