Cellular and Molecular Bioengineering

, Volume 2, Issue 2, pp 190–199 | Cite as

In vivo Multimotor Force–Velocity Curves by Tracking and Sizing Sub-Diffraction Limited Vesicles

  • Yuri Shtridelman
  • George M. Holzwarth
  • Clayton T. Bauer
  • Natalie R. Gassman
  • David A. DeWitt
  • Jed C. MacoskoEmail author


Determining in vivo force–velocity relationships of motor proteins is a critical step toward clarifying how they accomplish intracellular transport. We show that in vivo force–velocity curves corresponding to an estimated 1, 2, and 3 motors-per-vesicle can be constructed by tracking and sizing transported vesicles. The force range for these curves would normally be constrained by diffraction limited diameter measurements. However, we present a new method that uses the image intensity obtained with differential interference contrast microscopy as a proxy for vesicle diameters smaller than the diffraction limit. We calibrate this novel sizing method in vitro with polystyrene microsphere standards and apply it in vivo to vesicles. The resulting diameter vs. velocity data for large, small, and sub-diffraction limited vesicles is used to construct force–velocity curves that extend the force range of our previous curves. These extended 1-, 2-, and 3-motor in vivo curves qualitatively agree with a simple model of load sharing for motors that jointly transport a single vesicle.


Processive molecular motors Cooperative fast vesicle transport Anterograde retrograde traffic Intracellular motion DIC microscopy Kinesin Dyenin Myosin Particle tracking 



We thank Keith Bonin for helpful discussions for input at various stages. This work was supported by a start-up grant by Wake Forest University to JCM, an NIH grant (NS-053493) to GMH and by an NIH grant (AG-020996) to DAD.

Supplementary material

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Supplementary material 1 (DOC 36 kb)
12195_2009_64_MOESM2_ESM.avi (50 mb)
Supplementary material 2 (AVI 51209 kb)

Supplementary material 3 (AVI 9266 kb)


  1. 1.
    Barkus, R.V., et al., Identification of an axonal kinesin-3 motor for fast anterograde vesicle transport that facilitates retrograde transport of neuropeptides. Mol Biol Cell, 2008. 19(1): p. 274-83.CrossRefGoogle Scholar
  2. 2.
    Bausch, A.R., W. Moller, and E. Sackmann, Measurement of local viscoelasticity and forces in living cells by magnetic tweezers. Biophys J, 1999. 76(1 Pt 1): p. 573-9.CrossRefGoogle Scholar
  3. 3.
    Bednarski, E., C.E. Ribak, and G. Lynch, Suppression of cathepsins B and L causes a proliferation of lysosomes and the formation of meganeurites in hippocampus. J Neurosci, 1997. 17(11): p. 4006-21.Google Scholar
  4. 4.
    Brady, S.T., R.J. Lasek, and R.D. Allen, Fast axonal transport in extruded axoplasm from squid giant axon. Science, 1982. 218(4577): p. 1129-31.CrossRefGoogle Scholar
  5. 5.
    Breuer, A.C., et al., Fast axonal transport in amyotrophic lateral sclerosis: an intra-axonal organelle traffic analysis. Neurology, 1987. 37(5): p. 738-48.Google Scholar
  6. 6.
    Clemen, A.E., et al., Force-dependent stepping kinetics of myosin-V. Biophys J, 2005. 88(6): p. 4402-10.CrossRefGoogle Scholar
  7. 7.
    de Vries, A.H., et al., Micro magnetic tweezers for nanomanipulation inside live cells. Biophys J, 2005. 88(3): p. 2137-44.CrossRefGoogle Scholar
  8. 8.
    Foo, J.J., K.K. Liu, and V. Chan, Viscous drag of deformed vesicles in optical trap: experiments and simulations. AIChE Journal, 2004. 50(1): p. 249 - 254.CrossRefGoogle Scholar
  9. 9.
    Friberg, H., et al., Cyclosporin A, but not FK 506, protects mitochondria and neurons against hypoglycemic damage and implicates the mitochondrial permeability transition in cell death. J Neurosci, 1998. 18(14): p. 5151-9.Google Scholar
  10. 10.
    Gagliano, J., et al. Kinesin velocity increases with the number of motors pulling a viscoelastic load (submitted).Google Scholar
  11. 11.
    Gennerich, A. and D. Schild, Finite-particle tracking reveals submicroscopic-size changes of mitochondria during transport in mitral cell dendrites. Phys Biol, 2006. 3(1): p. 45-53.CrossRefGoogle Scholar
  12. 12.
    Grallert, A., et al., In vivo movement of the type V myosin Myo52 requires dimerisation but is independent of the neck domain. J Cell Sci, 2007. 120(Pt 23): p. 4093-8.CrossRefGoogle Scholar
  13. 13.
    Hill, D. B., Changes in the number of molecular motors driving vesicle transport in PC12, Physics Ph.D. dissertation. Wake Forest University, Winston-Salem, 2003Google Scholar
  14. 14.
    Hill, D.B., et al., Fast vesicle transport in PC12 neurites: velocities and forces. Eur Biophys J, 2004. 33(7): p. 623-32.CrossRefGoogle Scholar
  15. 15.
    Hirakawa, E., H. Higuchi, and Y.Y. Toyoshima, Processive movement of single 22S dynein molecules occurs only at low ATP concentrations. Proc Natl Acad Sci U S A, 2000. 97(6): p. 2533-7.CrossRefGoogle Scholar
  16. 16.
    Hirokawa, N., et al., Kinesin associates with anterogradely transported membranous organelles in vivo. J. Cell Biol., 1991. 114: p. 295–302.CrossRefGoogle Scholar
  17. 17.
    Hunt, A.J., F. Gittes, and J. Howard, The force exerted by a single kinesin molecule against a viscous load. Biophys J, 1994. 67: p. 766-781.CrossRefGoogle Scholar
  18. 18.
    Jasinski, A., A. Gorbman, and T.J. Hara, Rate of movement and redistribution of stainable neurosecretory granules in hypothalamic neurons. Science, 1966. 154(750): p. 776-8.CrossRefGoogle Scholar
  19. 19.
    Kaether, C., P. Skehel, and C.G. Dotti, Axonal Membrane Proteins are Transported in Distinct Carriers: A Two-Color Video Microscopy Study in Cultured Hippocampal Neurons. Mol Biol Cell, 2000. 11: p. 1213–1224.Google Scholar
  20. 20.
    Kawaguchi, K. and S. Ishiwata, Temperature dependence of force, velocity, and processivity of single kinesin molecules. Biochem Biophys Res Commun, 2000. 272(3): p. 895-9.CrossRefGoogle Scholar
  21. 21.
    Kawaguchi, K. and S.c. Ishiwata, Temperature dependence of force, velocity, and processivity of single kinesin molecules. Biochem. Biophys. Res. Commun., 2000. 272: p. 895-899.CrossRefGoogle Scholar
  22. 22.
    Kidwai, A.M. and S. Ochs, Components of fast and slow phases of axoplasmic flow. J Neurochem, 1969. 16(7): p. 1105-12.CrossRefGoogle Scholar
  23. 23.
    Kojima, H., et al., Mechanics of Single Kinesin Molecules Measured by Optical Trapping Nanometry. Biophys. J., 1997. 73(4): p. 2012-2022.CrossRefGoogle Scholar
  24. 24.
    Kural, C., et al., Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement? Science, 2005. 308(5727): p. 1469-72.CrossRefGoogle Scholar
  25. 25.
    Laib, J.A., et al., The reciprocal coordination and mechanics of molecular motors in living cells. Proc Natl Acad Sci U S A, 2009. 106(9): p. 3190-5.CrossRefGoogle Scholar
  26. 26.
    Levi, V., et al., Organelle transport along microtubules in Xenopus melanophores: evidence for cooperation between multiple motors. Biophys J, 2006. 90(1): p. 318-27.CrossRefGoogle Scholar
  27. 27.
    Ligon, L.A., et al., A direct interaction between cytoplasmic dynein and kinesin I may coordinate motor activity. J. Biol. Chem., 2004. 279(18): p. 19201-19208.CrossRefGoogle Scholar
  28. 28.
    Luby-Phelps, K., Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int Rev Cytol, 2000. 192: p. 189-221.CrossRefGoogle Scholar
  29. 29.
    Macosko, J.C., et al., Fewer active motors per vesicle may explain slowed vesicle transport in chick motoneurons after three days in vitro. Brain Res., 2008. 1211: p. 6-12.CrossRefGoogle Scholar
  30. 30.
    Mallik, R., et al., Cytoplasmic dynein functions as a gear in response to load. Nature, 2004. 427(6975): p. 649-52.CrossRefGoogle Scholar
  31. 31.
    Martin, E.J., et al., Analysis of Huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons. J Comp Neurol, 1999. 403(4): p. 421-30.CrossRefGoogle Scholar
  32. 32.
    Martinez, J.E., et al., On the use of in vivo cargo velocity as a biophysical marker. Biochem Biophys Res Commun, 2007. 353(3): p. 835-40.CrossRefGoogle Scholar
  33. 33.
    Mehta, A.D., et al., Myosin-V is a processive actin-based motor. Nature, 1999. 400(6744): p. 590-3.CrossRefGoogle Scholar
  34. 34.
    Meyhofer, E. and J. Howard, The force generated by a single kinesin molecule against an elastic load. Proc Natl Acad Sci U S A, 1995. 92(2): p. 574-8.CrossRefGoogle Scholar
  35. 35.
    Moreno, S., R. Nardacci, and M.P. Ceru, Regional and ultrastructural immunolocalization of copper-zinc superoxide dismutase in rat central nervous system. J Histochem Cytochem, 1997. 45(12): p. 1611-22.Google Scholar
  36. 36.
    Muller, M.J., S. Klumpp, and R. Lipowsky, Tug-of-war as a cooperative mechanism for bidirectional cargo transport by molecular motors. Proc Natl Acad Sci U S A, 2008. 105(12): p. 4609-14.CrossRefGoogle Scholar
  37. 37.
    Nan, X., P.A. Sims, and X.S. Xie, Organelle tracking in a living cell with microsecond time resolution and nanometer spatial precision. Chemphyschem, 2008. 9(5): p. 707-12.CrossRefGoogle Scholar
  38. 38.
    Pilling, A.D., et al., Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons. Mol Biol Cell, 2006. 17(4): p. 2057-68.CrossRefGoogle Scholar
  39. 39.
    Schnapp, B.J. and T.S. Reese, Dynein is the motor for retrograde axonal transport of organelles. Proc Natl Acad Sci U S A, 1989. 86(5): p. 1548-52.CrossRefGoogle Scholar
  40. 40.
    Shingyoji, C., et al., Dynein arms are oscillating force generators. Nature, 1998. 393(6686): p. 711-4.CrossRefGoogle Scholar
  41. 41.
    Shtridelman, Y., et al., Force–velocity curves of motor proteins cooperating in vivo. Cell Biochem. Biophys., 52, 19–29, 2008.CrossRefGoogle Scholar
  42. 42.
    Svoboda, K. and S.M. Block, Force and velocity measured for single kinesin molecules. Cell, 1994. 77(5): p. 773-84.CrossRefGoogle Scholar
  43. 43.
    Toba, S., et al., Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein. Proc Natl Acad Sci U S A, 2006. 103(15): p. 5741-5.CrossRefGoogle Scholar
  44. 44.
    Uemura, S., et al., Mechanochemical coupling of two substeps in a single myosin V motor. Nat Struct Mol Biol, 2004. 11(9): p. 877-83.CrossRefGoogle Scholar
  45. 45.
    Uyeda, T.Q., et al., Quantized velocities at low myosin densities in an in vitro motility assay. Nature, 1991. 352(6333): p. 307-11.CrossRefGoogle Scholar
  46. 46.
    Visscher, K., M.J. Schnitzer, and S.M. Block, Single kinesin molecules studied with a molecular force clamp. Nature, 1999. 400: p. 184-189.CrossRefGoogle Scholar
  47. 47.
    Welte, M.A., Bidirectional transport along microtubules. Curr Biol, 2004. 14(13): p. R525-37.CrossRefGoogle Scholar
  48. 48.
    Zahn, T.R., et al., Dense core vesicle dynamics in Caenorhabditis elegans neurons and the role of kinesin UNC-104. Traffic, 2004. 5(7): p. 544-59.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2009

Authors and Affiliations

  • Yuri Shtridelman
    • 1
  • George M. Holzwarth
    • 1
  • Clayton T. Bauer
    • 1
  • Natalie R. Gassman
    • 1
  • David A. DeWitt
    • 2
  • Jed C. Macosko
    • 1
    Email author
  1. 1.Department of PhysicsWake Forest UniversityWinston-SalemUSA
  2. 2.Department of Biology and ChemistryLiberty UniversityLynchburgUSA

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