An improved optical tweezers assay for measuring the force generation of single kinesin molecules

Matthew P. Nicholas, Lu Rao, Arne Gennerich

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560- GFP) in Escherichia coli, purifying it, and studying its force generation in an optical tweezers microscope. We also provide detailed instructions on proper alignment and calibration of an optical trapping microscope. These methods provide a foundation for a variety of similar experiments.

Original languageEnglish (US)
Pages (from-to)171-246
Number of pages76
JournalMethods in molecular biology (Clifton, N.J.)
Volume1136
DOIs
StatePublished - 2014
Externally publishedYes

Fingerprint

Optical Tweezers
Kinesin
Chromosome Positioning
Cytoplasmic Dyneins
Mitosis
Microtubules
Cell Division
Calibration
Escherichia coli
Proteins

Keywords

  • Escherichia coli
  • Force measurement
  • K560
  • Kinesin
  • Microtubules
  • Molecular motors
  • Optical trap alignment and calibration
  • Optical trapping
  • Optical tweezers
  • Protein purification
  • Pyranose oxidase
  • Single-molecule assays

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Medicine(all)

Cite this

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abstract = "Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560- GFP) in Escherichia coli, purifying it, and studying its force generation in an optical tweezers microscope. We also provide detailed instructions on proper alignment and calibration of an optical trapping microscope. These methods provide a foundation for a variety of similar experiments.",
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