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Force on Single Actin Filaments in a Motility Assay Measured with an Optical Trap

  • R. M. Simmonst
  • J. T. Finer
  • H. M. Warrick
  • B. Kralik
  • S. Chu
  • J. A. Spudich
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 332)

Abstract

We have used an optical trap to measure or exert a force on single actin filaments via the attachment of polystyrene beads which were coated with NEM-modified HMM. In the simplest experiment, beads were attached to rhodamine phalloidin labelled actin filaments and observed to move on an HMM coated surface in the presence of ATP. Moving beads were steered into the vicinity of the trap using a PZT operated microscope stage. The minimum force needed to stop a moving bead was measured by lowering the trap strength until the bead resumed movement. By aligning the optical trap with the centre of a quadrant detector placed in an image plane of the microscope, it was possible to measure the force exerted on a filament by measuring the displacement of the bead position from the centre of the trap. In each of these experiments, the trap was calibrated by applying a Stokes force to a bead in free solution. The characteristics of the trap were studied, and the displacement of the bead from the centre of the trap was shown to be directly proportional to the applied force over a large part of the total range of the trap. The compliance of the trap could be substantially reduced by the use of feedback control to deflect the laser beam via an acousto-optic modulator. The advantages and limitations of this technique will be discussed.

Keywords

Actin Filament Polystyrene Bead Motility Assay Optical Trap Quadrant Detector 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 1993

Authors and Affiliations

  • R. M. Simmonst
    • 1
  • J. T. Finer
    • 1
  • H. M. Warrick
    • 2
  • B. Kralik
    • 2
  • S. Chu
    • 3
  • J. A. Spudich
    • 3
  1. 1.MRC Muscle and Motility Unit Kings’s College LondonLondonUK
  2. 2.Department of BiochemistryStanford UniversityStanfordUSA
  3. 3.Department of PhysicsStanford UniversityStanfordUSA

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