Supplemental Figure S1 (Adobe PDF) -
Fig. S1. ALCAM-mediated adhesion probed by a ligand-coated, bead-modified AFM cantilever. (A) 10 pN/nm cantilevers (MSCT; Veeco Instruments, Santa Barbara, CA) with a 10 μm glass bead glued onto the tip were obtained from Novascan (Ames, IA). Individual cantilevers were calibrated using the thermal oscillation method. The beads were coated with streptavidin by the manufacturer and were further modified with biotinylated goat anti-human Fc-(Fab′)2 fragments and subsequently with purified recombinant ALCAM-Fc or CD6-Fc. The ALCAM- or CD6-coated cantilevers were used to probe the interaction with ALCAM expressed at the cell membrane of K562-ALCAM cells seeded on a poly-L-lysine-coated glass coverslip. Force measurements were performed on at least ten K562 cells in medium A. The interaction time between the bead and the cell was set to 3 seconds at a contact force of ∼500 pN. (B) Single-molecule force measurements. Histograms show the force distributions obtained by analyzing the final rupture events under two different pulling conditions. At a pulling velocity of 2.5 μm/second the mean rupture force and loading rates (± s.e.m; n>75) were 22±1 pN at 294±27 pN/second for the ALCAM-ALCAM bond and 28±1 pN at 331±30 pN/second for ALCAM-CD6. At a pulling velocity of 12.5 μm/second this amounted to 54±1 pN at 1473±135 pN/second for ALCAM-ALCAM and to 61±2 pN at 1659±152 pN/second for ALCAM-CD6. Although the mean loading rates obtained for both pulling conditions did not significantly differ, the ALCAM-CD6 bond ruptured at significantly higher forces than the homotypic interaction in both cases (P<0.05). A single Gaussian function (solid lines) could be fitted to the force distributions which in all cases accounted for >85% of the events, indicating that predominantly single-molecule rupture events were analyzed, see main text. (C) Specificity of the interactions. Analyses of the relative work of de-adhesion comparing the situation before treatment (dashed bars), with that after a 30 minute incubation with the ALCAM function blocking mAb AZNL50 (open bars). Both the homo- and heterotypic ALCAM-mediated interactions could be blocked, indicating that the interactions were indeed ALCAM-specific. At least 25 traces per cell were analyzed per condition (medium condition set to 100%; error bars represent s.e.m.; * indicates significance to P< 0.05.) (D) Bead-modified vs cell-functionalized cantilevers. Comparison of the mean rupture forces determined using bead-modified AFM cantilevers (stars, ALCAM-ALCAM; triangles, ALCAM-CD6; ± s.d.) as a function of the loading rate (±s.e.m.) and the data obtained using cell-functionalized cantilevers represented by the Bell model fit results taken from Fig. 5A. The data obtained using the bead-modified AFM cantilevers are in good agreement with the data obtained using the cell-functionalized AFM cantilevers.
