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 single-molecule 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 density-dependent 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.
Rock
R S
RS
Department of Biochemistry, Stanford University School of Medicine, Beckman Center B405, Stanford, California 94305-5307, USA.
Rief
M
M
Mehta
A D
AD
Spudich
J A
JA
D008841:Actin Cytoskeleton / Q000502:physiology; D001703:Biophysics / Q000379:methods*; D002148:Calmodulin-Binding Proteins / Q000502:physiology*; D020409:Molecular Motor Proteins / Q000502:physiology*; D009068:Movement / Q000502:physiology*; D018994:Myosin Light Chains / Q000502:physiology*; D024701:Myosin Type V; D009419:Nerve Tissue Proteins / Q000502:physiology*; D055095:Optics and Photonics
D002148:Calmodulin-Binding Proteins; D020409:Molecular Motor Proteins; D018994:Myosin Light Chains; D009419:Nerve Tissue Proteins; C078829:chicken brain myosin-V p190; D024701:Myosin Type V