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High-Resolution Single-Molecule Kinesin Assays at kHz Frame Rates

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Molecular Motors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1805))

Abstract

This chapter describes methods for high-speed, unloaded, in vitro single-molecule kinesin tracking experiments. Instructions are presented for constructing a total internal reflection dark-field microscope (TIRDFM) and labeling motors with gold nanoparticles. An AMP-PNP unlocking assay is introduced as a specialized means of capturing processive events in a reduced field of view. Finally, step-finding tools for analyzing high frame-rate tracking data are described.

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References

  1. Hackney DD (1994) Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis. Proc Natl Acad Sci U S A 91(15):6865–6869

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Yildiz A, Tomishige M, Vale RD (2004) Kinesin walks hand-over-hand. Science 303:676–678

    Article  CAS  PubMed  Google Scholar 

  3. Hancock WO, Howard J (1999) Kinesin’s processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains. Proc Natl Acad Sci U S A 96(23):13147–13152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Coy DL, Wagenbach M, Howard J (1999) Kinesin takes one 8-nm step for each ATP that it hydrolyzes. J Biol Chem 274(6):3667–3671

    Article  CAS  PubMed  Google Scholar 

  5. Schnitzer MJ, Block SM (1997) Kinesin hydrolyses one ATP per 8-nm step. Nature 388(6640):386–390

    Article  CAS  PubMed  Google Scholar 

  6. Block SM (2007) Kinesin motor mechanics: binding, stepping, tracking, gating, and limping. Biophys J 92(9):2986–2995

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Carter NJ, Cross RA (2005) Mechanics of the kinesin step. Nature 435(7040):308–312

    Article  CAS  PubMed  Google Scholar 

  8. Toprak E, Yildiz A, Tonks M, Rosenfeld SS, Selvin PR (2009) Why kinesin is so processive. Proc Natl Acad Sci U S A 106(31):12717–12722

    Article  PubMed  PubMed Central  Google Scholar 

  9. Andrecka J et al (2015) Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy. elife 4:e05413

    Article  PubMed Central  Google Scholar 

  10. Ortega Arroyo J et al (2014) Label-free, all-optical detection, imaging, and tracking of a single protein. Nano Lett 14:2065–2070

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dunn AR, Chuan P, Bryant Z, Spudich JA (2010) Contribution of the myosin VI tail domain to processive stepping and intramolecular tension sensing. Proc Natl Acad Sci U S A 107(17):7746–7750

    Article  PubMed  PubMed Central  Google Scholar 

  12. Mickolajczyk KJ, Deffenbaugh NC, Ortega Arroyo J, Andrecka J, Kukura P, Hancock WO (2015) Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle. Proc Natl Acad Sci U S A 112(52):E7186–E7193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Nan X, Sims PA, Xie XS (2008) Organelle tracking in a living cell with microsecond time resolution and nanometer spatial precision. ChemPhysChem 9(5):707–712

    Article  CAS  PubMed  Google Scholar 

  14. Ortega-Arroyo J, Kukura P (2012) Interferometric scattering microscopy (iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopy. Phys Chem Chem Phys 14(45):15625–15636

    Article  CAS  PubMed  Google Scholar 

  15. Schneider R, Glaser T, Berndt M, Diez S (2013) Using a quartz paraboloid for versatile wide- field TIR microscopy with sub-nanometer localization accuracy. Opt Express 21(3):686–689

    Article  CAS  Google Scholar 

  16. Sowa Y, Steel BC, Berry RM (2010) A simple backscattering microscope for fast tracking of biological molecules. Rev Sci Instrum 81(11):113704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ueno H et al (2010) Simple dark-field microscopy with nanometer spatial precision and microsecond temporal resolution. Biophys J 98(9):2014–2023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Dunn AR, Spudich JA (2007) Dynamics of the unbound head during myosin V processive translocation. Nat Struct Mol Biol 14(3):246–248

    Article  CAS  PubMed  Google Scholar 

  19. Braslavsky I et al (2001) Objective-type dark-field illumination for scattering from microbeads. Appl Opt 40(31):5650–5657

    Article  CAS  PubMed  Google Scholar 

  20. Yasuda R, Noji H, Yoshida M, Kinosita K, Itoh H (2001) Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase. Nature 410(6831):898–904

    Article  CAS  PubMed  Google Scholar 

  21. Chen G-Y, Mickolajczyk KJ, Hancock WO (2016) The kinesin-5 chemomechanical cycle is dominated by a two-heads-bound state. J Biol Chem 291(39):20283–20294

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Mickolajczyk KJ, Hancock WO (2017) Kinesin processivity is determined by a kinetic race from a vulnerable one-head-bound state. Biophys J 112(12):2615–2623

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Axelrod D, Burghardt TP, Thompson NL (1984) Total internal reflection fluorescence. Annu Rev Biophys Bioeng 13:247–268

    Article  CAS  PubMed  Google Scholar 

  24. Tokunaga M, Kitamura K, Saito K, Iwane a H, Yanagida T (1997) Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy. Biochem Biophys Res Commun 235(1):47–53

    Article  CAS  PubMed  Google Scholar 

  25. Chen G-Y, Arginteanu DFJ, Hancock WO (2015) Processivity of the kinesin-2 KIF3A results from rear head gating and not front head gating. J Biol Chem 290(16):10274–10294

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Schnapp BJ, Crise B, Sheetz MP, Reese TS, Khan S (1990) Delayed start-up of kinesin-driven microtubule gliding following inhibition by adenosine 5′-[beta,gamma-imido]triphosphate. Proc Natl Acad Sci U S A 87(24):10053–10057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Berliner E, Young EC, Anderson K, Mahtani HK, Gelles J (1995) Failure of a single-headed kinesin to track parallel to microtubule protofilaments. Nature 373:718–721

    Article  CAS  PubMed  Google Scholar 

  28. Guydosh NR, Block SM (2009) Direct observation of the binding state of the kinesin head to the microtubule. Nature 461(7260):125–128

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mori T, Vale RD, Tomishige M (2007) How kinesin waits between steps. Nature 450(7170):750–754

    Article  CAS  PubMed  Google Scholar 

  30. Verbrugge S, Lansky Z, Peterman EJG (2009) Kinesin’ s step dissected with single-motor FRET. Proc Natl Acad Sci U S A 106(42):17741–17746

    Article  PubMed  PubMed Central  Google Scholar 

  31. Farrell CM, Mackey AT, Klumpp LM, Gilbert SP (2002) The role of ATP hydrolysis for kinesin processivity. J Biol Chem 277(19):17079–17087

    Article  CAS  PubMed  Google Scholar 

  32. Uppalapati M, Huang Y, Shastry S, Jackson TN, Hancock WO (2009) Microtubule motors in microfluidics. Methods in bioengineering: microfabrication and microfluidics, pp 311–337

    Google Scholar 

  33. Larson J et al (2014) Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope. Nat Protoc 9(10):2317–2328

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Friedman LJ, Chung J, Gelles J (2006) Viewing dynamic assembly of molecular complexes by multi-wavelength single-molecule fluorescence. Biophys J 91(3):1023–1031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Block SM, Goldstein LS, Schnapp BJ (1990) Bead movement by single kinesin molecules studied with optical tweezers. Nature 348(6299):348–352

    Article  CAS  PubMed  Google Scholar 

  36. Ozeki T et al (2009) Surface-bound casein modulates the adsorption and activity of kinesin on SiO2 surfaces. Biophys J 96(8):3305–3318

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yildiz A (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300(5628):2061–2065

    Article  CAS  PubMed  Google Scholar 

  38. Ruhnow F, Zwicker D, Diez S (2011) Tracking single particles and elongated filaments with nanometer precision. Biophys J 100(11):2820–2828

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chen Y, Deffenbaugh NC, Anderson CT, Hancock WO (2014) Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex. Mol Biol Cell 25(22):3630–3642

    Article  PubMed  PubMed Central  Google Scholar 

  40. Kerssemakers JWJ et al (2006) Assembly dynamics of microtubules at molecular resolution. Nature 442(7103):709–712

    Article  CAS  PubMed  Google Scholar 

  41. Shastry S, Hancock WO (2010) Neck linker length determines the degree of processivity in kinesin-1 and kinesin-2 motors. Curr Biol 20(10):939–943

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Shastry S, Hancock WO (2011) Interhead tension determines processivity across diverse N-terminal kinesins. Proc Natl Acad Sci U S A 108(39):16253–162588

    Article  PubMed  PubMed Central  Google Scholar 

  43. Cohn SA, Ingold AL, Scholey JM (1989) Quantitative analysis of sea urchin egg kinesin-driven microtubule motility. J Biol Chem 264(8):4290–4297

    PubMed  CAS  Google Scholar 

  44. Andreasson JO et al (2015) Examining kinesin processivity within a general gating framework. elife 4:e07403

    Article  PubMed Central  Google Scholar 

  45. Hancock WO, Howard J (1998) Processivity of the motor mrotein kinesin requires two heads. J Cell Biol 140(6):1395–1405

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Nara I, Ishiwata S (2006) Processivity of kinesin motility is enhanced on increasing temperature. Biophysics (Oxf) 2:13–21

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Biomedical Engineering Department, Pennsylvania State University, University Park, PA, USA

    Keith J. Mickolajczyk & William O. Hancock

Authors
  1. Keith J. Mickolajczyk
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  2. William O. Hancock
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Corresponding author

Correspondence to William O. Hancock .

Editor information

Editors and Affiliations

  1. National Museum of Natural History, CNRS UMR7196 / INSERM U1154, Paris, France

    Christophe Lavelle

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Mickolajczyk, K.J., Hancock, W.O. (2018). High-Resolution Single-Molecule Kinesin Assays at kHz Frame Rates. In: Lavelle, C. (eds) Molecular Motors. Methods in Molecular Biology, vol 1805. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8556-2_7

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  • DOI: https://doi.org/10.1007/978-1-4939-8556-2_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8554-8

  • Online ISBN: 978-1-4939-8556-2

  • eBook Packages: Springer Protocols

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