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An Improved Optical Tweezers Assay for Measuring the Force Generation of Single Kinesin Molecules

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Mitosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1136))

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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Acknowledgments

We thank Joshua Shaevitz, Michael Diehl, Erik Schäffer, and Kenneth Jamison for helpful discussions on optical trapping, Marko Swoboda for helpful communications regarding the use of pyranose oxidase in the oxygen scavenging system, and Laura E.K. Nicholas for assistance with photography and figure illustration. The authors are supported by the National Institutes of Health grant R01GM098469. M.P.N. received support from the NIH-funded Medical Scientist Training and Molecular Biophysics Training programs (NIH grants T32GM007288 and T32GM008572, respectively) at the Albert Einstein College of Medicine.

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  1. Department of Anatomy and Structural Biology, Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA

    Matthew P. Nicholas, Lu Rao & Arne Gennerich

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  1. Matthew P. Nicholas
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  2. Lu Rao
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  3. Arne Gennerich
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Correspondence to Arne Gennerich .

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  1. Dept Physiology Biophysics, Albert Einstein College of Medicine, Bronx, New York, USA

    David J. Sharp

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Nicholas, M.P., Rao, L., Gennerich, A. (2014). An Improved Optical Tweezers Assay for Measuring the Force Generation of Single Kinesin Molecules. In: Sharp, D. (eds) Mitosis. Methods in Molecular Biology, vol 1136. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0329-0_10

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  • DOI: https://doi.org/10.1007/978-1-4939-0329-0_10

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