Fig. 3. The actin cytoskeleton regulates ALCAM binding avidity. (A) Typical force-distance curves of the homotypic ALCAM-mediated interaction between a KG1a cell and an ALCAM–Fc-coated plate, before (medium) and after treatment with the actin cytoskeleton inhibitor CytD, and after a subsequent blocking step (CytD + mAb AZN-L50). For clarity, the traces are shown with an offset. The substrate retraction speed was set to 2.5 µm/second. The work needed to detach the cell from the substrate (shaded areas), typically between 1x10^–16 and 3x10^–16 J for untreated cells, was taken as a measure for overall cell adhesion. (B) Whole-cell analyses of the relative work of de-adhesion comparing the situation before treatment (medium) with that after incubation with the ALCAM-function-blocking mAb AZN-L50, or after incubation with CytD alone and followed by a subsequent AZN-L50 incubation. The relative work of de-adhesion was determined from at least 25 traces per cell per condition (medium condition set to 100%). It can be seen that CytD treatment upregulates overall cell adhesion and that this adhesion is ALCAM-specific. (C) Single-bond-level rupture-force analyses. In contrast to the overall cell adhesion, the single-bond rupture forces under these loading conditions were found to be insensitive to the various treatments (relative force-scale; n>30). Error bars represent s.e.m.; * indicates significance to P<0.05; n.s., not significant. Trends were reproducibly observed in three independent experiments.