Demonstration of catch bonds between an integrin and its ligand

Abstract

Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. How force regulates integrin-ligand dissociation is unclear. We used atomic force microscopy to measure the force-dependent lifetimes of single bonds between a fibronectin fragment and an integrin alpha(5)beta(1)-Fc fusion protein or membrane alpha(5)beta(1). Force prolonged bond lifetimes in the 10-30-pN range, a counterintuitive behavior called catch bonds. Changing cations from Ca(2+)/Mg(2+) to Mg(2+)/EGTA and to Mn(2+) caused longer lifetime in the same 10-30-pN catch bond region. A truncated alpha(5)beta(1) construct containing the headpiece but not the legs formed longer-lived catch bonds that were not affected by cation changes at forces <30 pN. Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range. Thus, catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension.

Figure 1.

AFM experiment. (A) AFM schematic (not to scale) is shown. 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. (B) Functionalization of AFM. Molecules depicted represent a composite of several adsorbed or captured on the cantilever tip or the Petri dish. Extended and bent α₅β₁-Fc, depicted as heterodimers of an α (light blue) and a β (red) subunit fused to an Fc (yellow), and trα₅β₁-Fc, consisting of the β propeller (teal) and thigh (dark green) domains of the α subunit as well as the βA (pink), hybrid (dark blue), and plexin/semaphorin/integrin (tan) domains of the β 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. FNIII_7–10 (purple) was adsorbed on the cantilever tip. In some control experiments, FNIII_7–10 was replaced by an anti-α₅β₁ mAb (P1D6; green). (C) Force-scan trace without adhesion. 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. (D) Force-scan trace with adhesion. The color codes are the same as those in C, which illustrates a contact cycle with binding and lifetime measurement. Petri dish retraction resulted in a tensile force indicating binding. 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.

Figure 2.

Binding specificity. (A and B) α₅β₁-Fc data are shown. (C and D) trα₅β₁-Fc data are shown. Frequencies of adhesion between 10 µg/ml FNIII_7–10 adsorbed on cantilever tips and 10 µg/ml α₅β₁-Fc (A) or trα₅β₁-Fc (C) captured by 15 or 100 µg/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 3–5 spots. Frequencies (measured with the same protocol as in A and C) of adhesion between 10 µg/ml FNIII_7–10 coated on cantilever tips and 10 µg/ml α₅β₁-Fc (B) or trα₅β₁-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 trα₅β₁-Fc–functionalized Petri dishes (open bars) and with those between FNIII_7–10-coated tips and GG-7–coated Petri dishes without incubating with trα₅β₁-Fc (hatched bars) in the indicated cation conditions.

Figure 3.

Lifetimes of FN–α₅β₁ bonds. (A–C) Plots of lifetime versus force of α₅β₁-Fc–functionalized Petri dish dissociating from FNIII_7–10-coated cantilever tips (circles) in Ca²⁺/Mg²⁺ (A), Mg²⁺/EGTA (B), and Mn²⁺ (C). (D) A qualitatively similar plot (triangles) of mα₅β₁ reconstituted into lipid bilayer dissociating from FNIII_7–10-coated cantilever tips in Mg²⁺/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–GG-7 dissociation instead of FNIII_7–10–α₅β₁-Fc dissociation. Also shown in C is a lifetime versus force plot (diamonds) of α₅β₁-Fc–functionalized Petri dish dissociating from BSA-coated cantilever tips measured in Mn²⁺. (E) Schematic of the molecular arrangement indicating possible dissociation loci between FNIII_7–10 and α₅β₁-Fc or between α₅β₁-Fc and GG-7. (F) Schematic of the molecular arrangement for experiments that measured the capturing strength of the Fc–GG-7 interaction. Error bars indicate mean ± SEM.

Figure 4.

Lifetimes of antibody–antigen bonds. (A and B) Plots of lifetime (mean ± SEM) versus force of interactions between α₅β₁-Fc–functionalized Petri dish and P1D6-coated cantilever tips (A) and between HFN7.1 captured by anti–mouse Fc antibody preadsorbed on Petri dish and FNIII_7–10-coated cantilever tips (B). Schematics depict the molecular arrangement for the experiments.

Figure 5.

Lifetimes of FN–trα₅β₁ bonds. (A–C) Plots of lifetime (mean ± SEM) versus force of trα₅β₁-Fc–functionalized Petri dish dissociating from FNIII_7–10-coated cantilever tips (circles) in Ca²⁺/Mg²⁺ (A), Mg²⁺/EGTA (B), and Mn²⁺ (C). Data from Fig. 3 (A–C) are replotted in A–C for comparison. The lifetime versus force curves of α₅β₁-Fc–functionalized Petri dish dissociating from FNIII_7–10-coated cantilever tips are shown as triangles, and α₅β₁-Fc–coated cantilever tips dissociating from GG-7–functionalized Petri dish are shown as squares. (D) Schematic of the molecular arrangement indicating possible dissociation loci between FNIII_7–10 and trα₅β₁-Fc or between trα₅β₁-Fc and GG-7.

Figure 6.

Effects of activating mAbs. (A–C) Lifetime (mean ± SEM) versus force plots (circles) of α₅β₁-Fc–functionalized Petri dish dissociating from FNIII_7–10-coated cantilever tips in Mn²⁺ and 10 µg/ml TS2/16 (A) or 12G10 (B) or trα₅β₁-Fc–functionalized Petri dish dissociating from FNIII_7–10-coated cantilever tips in Mn²⁺ and 10 µg/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 trα₅β₁-Fc–functionalized Petri dish dissociating from FNIII_7–10-coated cantilever tips are shown as triangles, and α₅β₁-Fc–coated cantilever tips dissociating from GG-7–functionalized Petri dish are shown as squares. (D) Schematic of the molecular arrangement indicating binding sites for TS2/16 and 12G10.