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.
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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).
Neuman KC, Nagy A (2008) Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods 5:491–505CrossRefGoogle Scholar
Gardner MK, Charlebois BD, Janosi IM et al (2011) Rapid microtubule self-assembly kinetics. Cell 146:582–592CrossRefGoogle Scholar
Kerssemakers JW, Munteanu EL, Laan L et al (2006) Assembly dynamics of microtubules at molecular resolution. Nature 442:709–712CrossRefGoogle Scholar
Cecconi C, Shank EA, Bustamante C et al (2005) Direct observation of the three-state folding of a single protein molecule. Science 309:2057–2060CrossRefGoogle Scholar
Mizuno D, Tardin C, Schmidt CF et al (2007) Nonequilibrium mechanics of active cytoskeletal networks. Science 315:370–373CrossRefGoogle Scholar
Guo M, Ehrlicher AJ, Jensen MH et al (2014) Probing the stochastic, motor-driven properties of the cytoplasm using force spectrum microscopy. Cell 158:822–832CrossRefGoogle Scholar
Tolic-Norrelykke SF, Schaffer E, Howard J et al (2006) Calibration of optical tweezers with positional detection in the back focal plane. Rev Sci Instrum 77:103101CrossRefGoogle Scholar
Nicholas MP, Rao L, Gennerich A (2014) An improved optical tweezers assay for measuring the force generation of single kinesin molecules. Methods Mol Biol 1136:171–246CrossRefGoogle Scholar
Leidel C, Longoria RA, Gutierrez FM et al (2012) Measuring molecular motor forces in vivo: implications for tug-of-war models of bidirectional transport. Biophys J 103:492–500CrossRefGoogle Scholar
Rai AK, Rai A, Ramaiya AJ et al (2013) Molecular adaptations allow dynein to generate large collective forces inside cells. Cell 152:172–182CrossRefGoogle Scholar
Jun Y, Tripathy SK, Narayanareddy BR et al (2014) Calibration of optical tweezers for in vivo force measurements: how do different approaches compare? Biophys J 107:1474–1484CrossRefGoogle Scholar
Mas J, Richardson AC, Reihani SN et al (2013) Quantitative determination of optical trapping strength and viscoelastic moduli inside living cells. Phys Biol 10:046006CrossRefGoogle Scholar
Hendricks AG, Holzbaur ELF, Goldman YE (2012) Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors. Proc Natl Acad Sci 109:18447–18452CrossRefGoogle Scholar
Blocker A, Severin FF, Burkhardt JK et al (1997) Molecular requirements for bi-directional movement of phagosomes along microtubules. J Cell Biol 137:113–129CrossRefGoogle Scholar
Blehm BH, Schroer TA, Trybus KM et al (2013) In vivo optical trapping indicates kinesin’s stall force is reduced by dynein during intracellular transport. Proc Natl Acad Sci U S A 110:3381–3386CrossRefGoogle Scholar