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Force-velocity relationship of single actin filament interacting with immobilised myosin measured by electromagnetic technique

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2 Author(s)
S. -J. P. Holohan ; Imperial Coll. London, UK ; S. B. Marston

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 μm diameter Dynabeads conjugated with gelsolin-G actin. These were introduced into a motility cell coated with 100 μg/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.

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IEE Proceedings - Nanobiotechnology  (Volume:152 ,  Issue: 3 )