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Measuring Molecular Forces Using Calibrated Optical Tweezers in Living Cells

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1486)

Abstract

Optical tweezers have been instrumental in uncovering the mechanisms motor proteins use to generate and react to force. While optical traps have primarily been applied to purified, in vitro systems, emerging methods enable measurements in living cells where the actively fluctuating, viscoelastic environment and varying refractive index complicate calibration of the instrument. Here, we describe techniques to calibrate optical traps in living cells using the forced response to sinusoidal oscillations and spontaneous fluctuations, and to measure the forces exerted by endogenous ensembles of kinesin and dynein motor proteins as they transport cargoes in the cell.

Key words

Optical trap Optical tweezers Kinesin Dynein Intracellular transport Microtubules Cell mechanics Live-cell assays Biological materials 

Notes

Acknowledgements

The authors thank Mr. Pritish Agarwal for developing custom software to control the optical trap, Mr. Pete Cainfrani for building the custom focus stabilization system, and Ms. Mariko Tokito for sharing her wealth of knowledge on cell culture and protein purification. This work was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant to AGH) and the National Institutes of Health (P01-GM087253 to YEG).

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of BioengineeringMcGill UniversityMontrealCanada
  2. 2.Pennsylvania Muscle Institute, Perelman School of MedicineUniversity of PennsylvaniaPhiladelphiaUSA
  3. 3.Department of Physiology, Perelman School of MedicineUniversity of PennsylvaniaPhiladelphiaUSA

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