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Single-molecule fluorescence to study molecular motors

  • Hyokeun Park (a1), Erdal Toprak (a2) and Paul R. Selvin (a2) (a3)
  • DOI:
  • Published online: 01 July 2007

Molecular motors, which use energy from ATP hydrolysis to take nanometer-scale steps with run-lengths on the order of micrometers, have important roles in areas such as transport and mitosis in living organisms. New techniques have recently been developed to measure these small movements at the single-molecule level. In particular, fluorescence imaging has contributed to the accurate measurement of this tiny movement. We introduce three single-molecule fluorescence imaging techniques which can find the position of a fluorophore with accuracy in the range of a few nanometers. These techniques are named after Hollywood animation characters: Fluorescence Imaging with One Nanometer Accuracy (FIONA), Single-molecule High-REsolution Colocalization (SHREC), and Defocused Orientation and Position Imaging (DOPI). We explain new understanding of molecular motors obtained from measurements using these techniques.

Corresponding author
*Author for correspondence: Dr P. R. Selvin, Physics Department, 1110 West Green Street, Loomis Laboratory, University of Illinois, Urbana, IL 61801, USA. Tel.: 217-244-3371; Fax: 217-244-7559; E-mail:
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This list contains references from the content that can be linked to their source. For a full set of references and notes please see the PDF or HTML where available.

S. R. Adams , R. E. Campbell , L. A. Gross , B. R. Martin , G. K. Walkup , Y. Yao , J. Llopis & R. Y. Tsien (2002). New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. Journal of the American Chemical Society 124, 60636076.

C. L. Asbury , A. N. Fehr & S. M. Block (2003). Kinesin moves by an asymmetric hand-over hand mechanism. Science 302, 21302134.

K. B. Avraham , T. Hasson , T. Sobe , B. Balsara , J. R. Testa , A. B. Skvorak , C. C. Morton , N. G. Copeland & N. A. Jenkins (1997). Characterization of unconventional MYO6, the human homologue of the gene responsible for deafness in Snell's waltzer mice. Human Molecular Genetics 6, 12251231.

K. B. Avraham , T. Hasson , K. P. Steel , D. M. Kingsley , L. B. Russell , M. S. Mooseker , N. G. Copeland & N. A. Jenkins (1995). The mouse Snell's waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner ear hair cells. Nature Genetics 11, 369375.

A. Bahloul , G. Chevreux , A. L. Wells , D. Martin , J. Nolt , Z. Yang , L.-Q. Chen , N. Potier , A. Van Dorsselaer , S. Rosenfeld , A. Houdusse & H. L. Sweeney (2004). The unique insert in myosin VI is a structural calcium-calmodulin binding site. Proceedings of the National Academy of Sciences USA 101, 47874792.

H. Balci , T. Ha , H. L. Sweeney & P. R. Selvin (2005). Interhead distance measurements in myosin VI via SHRImP support a simplified hand-over-hand model. Biophysical Journal 89, 413417.

A. P. Bartko & R. M. Dickson (1999). Three-dimensional orientations of polymer-bound single molecules. Journal of Physical Chemistry B 103, 30533056.

J. S. Berg , B. C. Powell & R. E. Cheney (2001). A millennial myosin census. Molecular Biology of the Cell 12, 780794.

N. Bobroff (1986). Position measurement with a resolution and noise-limited instrument. Review of Scientific Instruments 57, 11521157.

M. Böhmer & J. Enderlein (2003). Orientation imaging of single molecules by wide-field epi-fluorescence microscopy. Journal of the Optical Society of America B 20, 554559.

S. Burgess , M. Walker , F. Wang , J. R. Sellers , H. D. White , P. J. Knight & J. Trinick (2002). The prepower stroke conformation of myosin V. Journal of Cell Biology 159, 983991.

F. Buss , G. Spudich & J. Kendrick-Jones (2004). Myosin VI: cellular functions and motor properties. Annual Review of Cell Developmental Biology 20, 649676.

M. K. Cheezum , W. F. Walker & W. H. Guilford (2001). Quantitative comparison of algorithms for tracking single fluorescent particles. Biophysical Journal 81, 23782388.

G. Chevreux , N. Potier , A. Van Dorsselaer , A. Bahloul , A. Houdusse , A. Wells & H. L. Sweeney (2005). Electrospray ionization mass spectrometry studies of noncovalent myosin VI complexes reveal a new specific calmodulin binding site. Journal of the American Society for Mass Spectrometry 16, 13671376.

L. S. Churchman , Z. Okten , R. S. Rock , J. F. Dawson & J. A. Spudich (2005). Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proceedings of the National Academy of Sciences USA 102, 14191423.

M. Dahan , S. Levi , C. Luccardini , P. Rostaing , B. Riveau & A. Triller (2003). Diffusion dynamics of glycine receptors revealed by single quantum dot tracking. Science 302, 442445.

J. Enderlein , E. Toprak & P. R. Selvin (2006). Polarization effect on position accuracy of fluorophore localization. Optics Express 14, 81118120.

J. N. Forkey , M. E. Quinlan , M. A. Shaw , J. E. Corrie & Y. E. Goldman (2003). Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization. Nature 422, 399404.

I. R. Gibbons , B. H. Gibbons , G. Mocz & D. J. Asai (1991). Multiple nucleotide-binding sites in the sequence of dynein beta heavy chain. Nature 352, 640643.

M. P. Gordon , T. Ha & P. R. Selvin (2004). Single-molecule high-resolution imaging with photobleaching. Proceedings of the National Academy of Sciences USA 101, 64626465.

T. Ha , T. Enderle , S. Chemla , R. Selvin & S. Weiss (1996). Single molecule dynamics studied by polarization modulation. Physical Review Letters 77, 39793982.

T. Hasson (2003). Myosin VI: two distinct roles in endocytosis. Journal of Cell Science 116, 34533461.

N. Hirokawa & R. Takemura (2004). Molecular motors in neuronal development, intracellular transport and diseases. Current Opinion in Neurobiology 14, 564573.

J. Hu , L. Li , W. Yang , L. Manna , L. Wang & A. P. Alivisatos (2001). Linearly polarized emission from colloidal semiconductor quantum rods. Science 292, 20602063.

W. Hua , J. Chung & J. Gelles (2002). Distinguishing inchworm and hand-over-hand processive kinesin movement by neck rotation measurements. Science 295, 844848.

J. D. Jaiswal & S. M. Simon (2007). Imaging single events at the cell membrane. Nature Chemical Biology 3, 9298.

K. Kaseda , H. Higuchi & K. Hirose (2003). Alternate fast and slow stepping of a heterodimeric kinesin molecule. Nature Cell Biology 5, 10791082.

K. A. Kellerman & K. G. Miller (1992). An unconventional myosin heavy chain gene from Drosophila melanogaster. Journal of Cell Biology 119, 823834.

S. J. King , M. Bonilla , M. E. Rodgers & T. A. Schroer (2002). Subunit organization in cytoplasmic dynein subcomplexes. Protein Science 11, 12391250.

K. Kinosita Jr., H. Itoh , S. Ishiwata , K. Hirano , T. Nishizaka & T. Hayakawa (1991). Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium. Journal of Cell Biology 115, 6773.

K. Kinosita Jr., R. Yasuda , H. Noji , S. Ishiwata & M. Yoshida (1998). F1-ATPase is a highly efficient molecular motor that rotates with discrete 120 degree steps. Cell 93, 2124.

T. Kon , T. Mogami , R. Ohkura , M. Nishiura & K. Sutoh (2005). ATP hydrolysis cycle-dependent tail motions in cytoplasmic dynein. Nature Structural & Molecular Biology 12, 513519.

M. P. Koonce (1997). Identification of a microtubule-binding domain in a cytoplasmic dynein heavy chain. Journal of Biological Chemistry 272, 1971419718.

C. Kural , H. Kim , S. Syed , G. Goshima , V. I. Gelfand & P. R. Selvin (2005). Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement? Science 308, 14691472.

I. Lister , S. Schmitz , M. Walker , J. Trinick , F. Buss , C. Veigel & J. Kendrick-Jones (2004). A monomeric myosin VI with a large working stroke. EMBO Journal 23, 17291738.

R. Mallik & S. P. Gross (2004). Molecular motors: strategies to get along. Current Biology 14, R971R982.

A. D. Mehta , R. S. Rock , M. Rief , J. A. Spudich , M. S. Mooseker & R. E. Cheney (1999). Myosin-V is a processive actin-based motor. Nature 400, 590593.

S. Melchionda , N. Ahituv , L. Bisceglia , T. Sobe , F. Glaser , R. Rabionet , M. L. Arbones , A. Notarangelo , E. D. Iorio , M. Carella , L. Zelante , X. Estivill , K. B. Avraham & P. Gasparini (2001). MYO6, the human homologue of the gene responsible for deafness in Snell's waltzer mice, is mutated in autosomal dominant nonsyndromic hearing loss. American Journal of Human Genetics 69, 635640.

J. Ménétrey , A. Bahloul , A. L. Wells , C. M. Yengo , C. A. Morris , H. L. Sweeney & A. Houdusse (2005). The structure of the myosin VI motor reveals the mechanism of directionality reversal. Nature 435, 779785.

L. S. Milescu , A. Yildiz , P. R. Selvin & F. Sachs (2006). Extracting dwell time sequences from processive molecular motor data. Biophysical Journal 91, 31353150.

K. E. Miller & M. P. Sheetz (2000). Characterization of myosin V binding to brain vesicles. Journal of Biological Chemistry 275, 25982606.

H. Millo , K. Leaper , V. Lazou & M. Bownes (2004). Myosin VI plays a role in cell-cell adhesion during epithelial morphogenesis. Mechanisms of Development 121, 13351351.

W. E. Moerner & D. P. Fromm (2003). Methods of single-molecule fluorescence spectroscopy and microscopy. Review of Scientific Instruments 74, 35973619.

X. Nan , P. A. Sims , P. Chen & X. S. Xie (2005). Observation of individual microtubule in living cells with endocytosed quantum dots. Journal of Physical Chemistry 109, 2422024224.

K. Ogawa (1991). Four ATP-binding sites in the midregion of the beta heavy chain of dynein. Nature 352, 643645.

Z. Okten , L. S. Churchman , R. S. Rock & J. A. Spudich (2004). Myosin VI walks hand-over-hand along actin. Nature Structural & Molecular Biology 11, 884887.

H. Park , G. Hanson , S. Duff & P. R. Selvin (2004). Nanometre localization of single ReAsH molecules. Journal of Microscopy 216, 199205.

H. Park , A. Li , L.-Q. Chen , A. Houdusse , P. R. Selvin & H. L. Sweeney (2007). The unique inset at the end of the myosin VI motor is the sole determinant of directionality. Proceedings of the National Academy of Sciences USA 104, 778783.

H. Park , B. Ramamurthy , D. Safer , M. Travaglia , L.-Q. Chen , C. Franzini-Armstrong , P. R. Selvin & H. L. Sweeney (2006). Full-length myosin VI dimerizes and moves processively along actin filaments upon monomer clustering. Molecular Cell 21, 331336.

B. M. Paschal & R. B. Vallee (1987). Retrograde transport by the microtubule-associated protein MAP 1C. Nature 330, 181183.

D. Patra , I. Gregor & J. Enderlein (2004). Image analysis of defocused single-molecule images for three-dimensional molecule orientation studies. Journal of Physical Chemistry A 108, 68366841.

E. J. G. Peterman , H. Sosa & W. E. Moerner (2004). Single-molecule fluorescence spectroscopy and microscopy of biomolecular motors. Annual Review of Physical Chemistry 55, 7996.

X. Qu , D. Wu , L. Mets & N. F. Scherer (2004). Nanometer-localized multiple single-molecule fluorescence microscopy. Proceedings of the National Academy of Sciences USA 101, 1129811303.

I. Rasnik , S. A. McKinney & T. Ha (2006). Nonblinking and long-lasting single molecule fluorescence imaging. Nature Methods 3, 891893.

S. L. Reck-Peterson , D. W. Provance , M. S. Mooseker & J. A. Mercer (2000). Review: class V myosins. Biochimica et Biophysica Acta 1496, 3651.

S. L. Reck-Peterson , A. Yildiz , A. P. Carter , A. Gennerich , N. Zhang & R. D. Vale (2006). Single-molecule analysis of dynein processivity and stepping behavior. Cell 126, 335348.

E. Reid , M. Kloos , A. Ashley-Koch , L. Hughes , S. Bevan , I. K. Svenson , F. L. Graham , P. C. Gaskell , A. Dearlove , M. A. Pericak-Vance , D. C. Rubinsztein & D. A. Marchuk (2002). A kinesin heavy chain (KIF5A) mutation in hereditary spastic paraplegia (SPG10). American Journal of Human Genetics. 71, 11891194.

R. S. Rock , B. Ramamurthy , A. R. Dunn , S. Beccafico , C. Morris , B. Spink , B. Rami , C. Franzini-Armstrong , J. A. Spudich & H. L. Sweeney (2005). A flexible domain is essential for the large step size and processivity of myosin VI. Molecular Cell 17, 603609.

R. S. Rock , S. E. Rice , A. L. Wells , T. J. Purcell , J. A. Spudich & H. L. Sweeney (2001). Myosin VI is a processive motor with a large step size. Proceedings of the National Academy of Sciences USA 98, 1365513659.

T. Sakamoto , I. Amitani , E. Yokota & T. Ando (2000). Direct observation of processive movement by individual myosin V molecules. Biochemical and Biophysical Research Communication 272, 586590.

W. S. Saunders , D. Koshland , D. Eshel , I. R. Gibbons & M. A. Hoyt (1995). Saccharomyces cerevisiae kinesin- and dynein-related proteins required for anaphase chromosome segregation. Journal of Cell Biology 128, 617624.

M. Schliwa & G. Woehlke (2003). Molecular motors. Nature 422, 759765.

M. J. Schnitzer & S. M. Block (1997). Kinesin hydrolyses one ATP per 8-nm step. Nature 388, 386390.

C. L. Schoch , J. R. Aist , O. C. Yoder & B. G. Turgeon (2003). A complete inventory of fungal kinesins in representative filamentous ascomycetes. Fungal Genetics and Biology 39, 115.

P. R. Selvin (2003). Lighting up single ion channels. Biophysical Journal 84, 12.

G. E. Snyder , T. Sakamoto , J. A. Hammer 3rd, J. R. Sellers & P. R. Selvin (2004). Nanometer localization of single green fluorescent proteins: evidence that myosin V walks hand-over-hand via telemark configuration. Biophysical Journal 87, 17761783.

J. A. Spudich (1994). How molecular motors work. Nature 372, 515518.

K. Svoboda , C. F. Schmidt , B. J. Schnapp & S. M. Block (1993). Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721727.

S. Syed , G. E. Snyder , C. Franzini-Armstrong , P. R. Selvin & Y. E. Goldman (2006). Simultaneous detection of the position and structural dynamics of myosin V. EMBO Journal 25, 17951803.

R. E. Thompson , D. R. Larson & W. W. Webb (2002). Precise nanometer localization analysis for individual fluorescent probes. Biophysical Journal 82, 27752783.

E. Toprak , J. Enderlein , S. Syed , S. A. McKinney , R. G. Petschek , T. Ha , Y. E. Goldman & P. R. Selvin (2006). Defocused orientation and position imaging (DOPI) of myosin V. Proceedings of the National Academy of Sciences USA 103, 64956499.

R. D. Vale (2003a). Myosin V motor proteins: marching stepwise towards a mechanism. Journal of Cell Biology 163, 445450.

R. D. Vale (2003b). The molecular motor toolbox for intracellular transport. Cell 112, 467480.

E. A. Vaisberg , M. P. Koonce & J. R. McIntosh (1993). Cytoplasmic dynein plays a role in mammalian mitotic spindle formation. Journal of Cell Biology 123, 849858.

R. B. Vallee , J. C. Williams , D. Varma & L. E. Barnhart (2004). Dynein: an ancient motor protein involved in multiple modes of transport. Journal of Neurobiology 58, 189200.

D. W. Warshaw , G. G. Kennedy , S. S. Work , E. B. Krementsova , S. Beck & K. M. Trybus (2005). Differential labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity. Biophysical Journal 88, L30L32.

A. Yildiz , J. N. Forkey , S. A. McKinney , T. Ha , Y. E. Goldman & P. R. Selvin (2003). Myosin V walks hand-over-hand: Single fluorophore imaging with 1·5 nm localization. Science 300, 20612065.

A. Yildiz , H. Park , D. Safer , Z. Yang , L.-Q. Chen , P. R. Selvin & H. L. Sweeney (2004a). Myosin VI steps via a hand-over-hand mechanism with its lever arm undergoing fluctuations when attached to actin. Journal of Biological Chemistry 279, 3722337226.

A. Yildiz , M. Tomishige , R. D. Vale & P. R. Selvin (2004b). Kinesin walks hand-over-hand. Science 303, 676678.

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