In Vitro Assays of Processive Myosin Motors

Myosin V is an actin-based motor thought to be involved in vesicle transport. Since the properties of such a motor may be expected to differ from those of muscle myosin II, we have examined myosin V-driven movement using a combination of gliding filament and optical trap assays to observe single molecules with high resolution. The results clearly demonstrate that brain myosin V is a highly efficient processive motor. In vitro motility assays at low myosin V densities reveal apparent singlemolecule supported movement. Processive stepping was also observed in optical trapping assays of myosin V-driven motion. Here the methods that were used to demonstrate the processivity of myosin V are described. These methods include densitydependent assays that eliminate the possibility of aggregation or chance colocalization of multiple motors being responsible for apparent single-molecule motility. Such assays will be useful tools for identifying other processive classes of myosins.