title: Force-velocity relationship of single actin filament interacting with immobilised myosin measured by electromagnetic technique.
abstract: The effect of applying an external load to actin filaments moving in the in vitro motility assay is studied. Bead-tailed actin filaments were made by polymerising actin onto 2.8 microm diameter Dynabeads conjugated with gelsolin-G actin. These were introduced into a motility cell coated with 100 microg/ml rabbit fast skeletal myosin in the presence of ATP and 0.5% methylcellulose. The motility cell was inserted between the pole-pieces of an electromagnet and the fluorescent beads and filaments were observed. The force-current relationship of the electromagnet was determined from the velocity of free beads in viscous solution and Stokes' equation. The magnet produced up to 6 pN force on the Dynabeads at 1 A. Many bead-tailed actin filaments stuck to the surface, but the beads that did move moved at the same speed as unloaded f-actin in the same cell. Bead-tailed filaments slowed down under an increasing magnetic load, eventually stalled and then slid backward under increasing load before detaching from the surface. Single-filament force-velocity curves were constructed and a stalling force of about 0.6 pN/mm of actin filament estimated.
authors.affiliation: Nation Heart and Lung Institute, Imperial College London, London SW3 6LY, UK.
authors.forename: S-J P
authors.initials: SJ
authors.lastname: Holohan
authors.forename: S B
authors.initials: SB
authors.lastname: Marston
mesh_terms: D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000818:Animals; D004548:Elasticity; D004574:Electromagnetic Fields; D008280:Magnetics; D008846:Micromanipulation / Q000295:instrumentation / Q000379:methods*; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000648:ultrastructure; D009218:Myosins / Q000737:chemistry* / Q000648:ultrastructure; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding; D011817:Rabbits; D013314:Stress, Mechanical
chemical_list: D020409:Molecular Motor Proteins; D009218:Myosins