Allophycocyanin

For characterization of the mCD1d–α-GalCer tetramer, cells isolated from various organs were resuspended in PBS staining buffer containing 2% BSA and 0.02% NaN₃. Cells were incubated for 15 min at 4°C with the blocking 2.4G2 anti-FcγR mAb and neutravidin (Molecular Probes) in a twofold excess of the neutravidin–PE contained in the amount of tetramer to be used for the staining. Neutravidin blocking was done to avoid any nonspecific binding of the neutravidin to biotin on the cells. Staining of FITC-, PE-, Cy-Chrome–, and allophycocyanin-conjugated mAbs was done simultaneously with the tetramer in PBS staining buffer at 23°C (room temperature) for 20 min. Cells were washed twice, and staining with biotinylated mAb was performed at 4°C for 20 min. After washing cells, tricolor-conjugated streptavidin was added as a secondary staining reagent for the biotinylated mAb and incubated for 15 min at 4°C, and cells were washed two times before analysis. mAbs used in this study include FITC-, Cy-Chrome–, or allophycocyanin-labeled anti–TCR-β clone H57-597, biotinylated or PE-labeled anti-NK1.1 clone PK136, Cy-Chrome–labeled anti-CD4 clone RM4-4, allophycocyanin-labeled anti-CD8α clone 53-6.7, FITC-labeled anti-CD5 clone 53-7.3, FITC-labeled anti-CD44 clone IM7, biotinylated anti-CD69 clone H1.2F3, FITC-labeled anti-Vβ2 clone B20.6, FITC-labeled anti-Vβ7 clone TR310, FITC-labeled anti-Vβ8.1/8.2 clone MR5-2, and FITC-labeled anti-Vβ12 clone MR11-1 (PharMingen). For intracellular staining, cells were incubated with blocking 2.4G2 anti-FcγR mAb and neutravidin (Molecular Probes) and then surface stained with TCR-β–FITC and either tetramer or anti-NK1.1–PE at 23°C. Cells were permeabilized using Cytofix/Cytoperm Plus™ (PharMingen) and stained using either FITC-labeled anti–IL-4 clone BVD4-1D11 or FITC-labeled anti–IFN-γ clone XMG1.2 (PharMingen) according to the manufacturer's protocol.

Recombinant α₅β₁-Fc and trα₅β₁-Fc chimeras were generated by CHOL761h cells that were transiently transfected with cDNA constructs encoding the human α₅ (Fitzgerald et al., 1987 (link)) and β₁ (Coe et al., 2001 (link)) subunits or trα₅ and trβ₁ subunits (Coe et al., 2001 (link)). Purified mα₅β₁ was obtained from Millipore. FNIII_7–10 with a biotin tag at the N terminus was produced using standard recombinant DNA techniques (Petrie et al., 2006 (link)). Anti-α₅β₁–blocking (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 (link); Sarangapani et al., 2004 (link)) built and calibrated in house. 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 (3–12 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 α₅β₁-Fc or trα₅β₁-Fc with FNIII_7–10 (Fig. 3 E and Fig. 5 D) or α₅β₁-Fc with P1D6 (Fig. 4 A, schematic), cantilever tips were incubated with 10–20 µg/ml FNIII_7–10 or P1D6 overnight at 4°C (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). The fragmented GG-7 (at coating concentration indicated in Fig. 2) was adsorbed on a small spot on a Petri dish by overnight incubation at 4°C. To capture trα₅β₁-Fc, the GG-7–precoated Petri dish was rinsed three times with TBS and incubated with 10 µg/ml trα₅β₁-Fc at the desired cation condition (Ca²⁺/Mg²⁺, Mg²⁺/EGTA, or Mn²⁺) 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 µg/ml HFN7.1, 1 mg/ml cyclo(-GRGDSP) (AnaSpec, Inc.), or 10 µg/ml TS2/16 or 12G10 (Fig. 6 D) were added to the buffer.
To measure the interaction of FNIII_7–10 with HFN7.1 (Fig. 4 B, schematic) or α₅β₁-Fc with GG-7 Fab (Fig. 3 F), FNIII_7–10 or α₅β₁-Fc was adsorbed on cantilevers and treated as described in the previous paragraph. 10 µg/ml goat anti–mouse Fc polyclonal antibody or 20 µg/ml fragmented GG-7 was adsorbed on a labeled spot on a Petri dish overnight at 4°C. To capture HFN7.1, the Petri dish was rinsed three times with TBS and incubated with 10 µg/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 FNIII_7–10 with mα₅β₁, cantilevers were coated with streptavidin at 50 µg/ml overnight at 4°C and further functionalized by incubation with 10 µl of 1 µg/ml FNIII_7–10 for 15 min at room temperature. mα₅β₁-incoporated lipid vesicle solution was prepared according to Muller et al. (1993) (link). 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 mα₅β₁, 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 37°C for 4 h. The resulting lipid vesicle solution was stored under argon at 4°C 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 100°C 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-µl drop of mα₅β₁-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 Mg²⁺, 2 mM EGTA, and 1% BSA. The mα₅β₁ bilayers that formed had low molecular densities to ensure their infrequent binding to the FNIII_7–10-coated cantilever tips, as required for measuring single bonds. Bilayers were immediately used in AFM experiments.
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 (0–5 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–D). 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 (5–70 pN) using a cantilever and a Petri dish in each experiment. Measurements from all of the experiments were pooled and binned. For example, for FN–α₅β₁-Fc–GG-7 interaction in 1 mM Ca²⁺/Mg²⁺ condition, 13 experiments were performed to acquire 846 lifetimes at forces ranging from 5–70 pN. These were segregated into 12 force bins, each of which spanned 5–6 pN and contained at least ∼50 lifetimes. Lifetimes were plotted versus force as mean ± SEM in Figs. 3–6. Plots of natural log (number of events with a lifetime >t) versus t were exemplified for the FN–α₅β₁-Fc–GG-7 bond in the three cation conditions (Fig. S5, A–C) and for the α₅β₁-Fc–GG-7 bond (Fig. S5 D).
Binding of 9EG7 to α₅β₁-Fc in different divalent cations was determined by flow cytometry. 125 µg (50 µl) 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 µg/ml for 30 min. 40 µl beads were washed three times in TBS, incubated with 20 µg/ml α₅β₁-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 µg/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, beads were analyzed by flow cytometry.