Figure 2.

The laser trap assay. To measure the force of binding to actin of unphosphorylated myosin, a single beam laser trap assay is used as follows: The laser trap is used to capture a polystyrene bead coated with N-ethylmaleimide modified myosin. Such myosin can attach to actin but does not cycle, so it is used to glue the fluorescently labeled actin filament. The actin is then brought in contact with the randomly adhered unphosphorylated myosin on a pedestal on the coverslip (A). After allowing time for binding, the pedestal is moved away from the laser trap at constant speed (B). Initially, the bead remains offset from the trap center. When the pulling force exerted by the trap exceeds the binding force of the unphosphorylated myosin molecules, the bead springs back into the trap center, its unloaded position. (C) The unbinding force is the product of the maximal distance between the bead and the trap center (Δd) by the trap stiffness. This force can be normalized by the number of myosin molecules estimated to interact with the actin filament.