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Force–Velocity Curves of Motor Proteins Cooperating In Vivo

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Abstract

Motor proteins convert chemical energy into work, thereby generating persistent motion of cellular and subcellular objects. The velocities of motor proteins as a function of opposing loads have been previously determined in vitro for single motors. These single molecule “force–velocity curves” have been useful for elucidating motor kinetics and for estimating motor performance under physiological loads due to, for example, the cytoplasmic drag force on transported organelles. Here we report force–velocity curves for single and multiple motors measured in vivo. Using motion enhanced differential interference contrast (MEDIC) movies of living NT2 (neuron-committed teratocarcinoma) cells at 37°C, three parameters were measured—velocity (v), radius (a), and effective cytoplasmic viscosity (η′)—as they applied to moving vesicles. These parameters were combined in Stokes’ equation, F = 6πaη′v, to determine the force, F, required to transport a single intracellular particle at velocity, v. In addition, the number of active motors was inferred from the multimodal pattern seen in a normalized velocity histogram. Using this inference, the resulting in vivo force–velocity curve for a single motor agrees with previously reported in vitro single motor force–velocity curves. Interestingly, however, the curves for two and three motors lie significantly higher in both measured velocity and computed force, which suggests that motors can work cooperatively to attain higher transport forces and velocities.

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Abbreviations

NT2:

Neuron-committed teratocarcinoma

DIC:

Differential interference contrast

MEDIC:

Motion enhanced DIC

References

  1. Koch, T., & Hollt, V. (2008). Role of receptor internalization in opioid tolerance and dependence. Pharmacology and Therapeutics, 117, 199–206.

    Article  PubMed  CAS  Google Scholar 

  2. Bie, B., & Pan, Z. Z. (2007). Trafficking of central opioid receptors and descending pain inhibition. Molecular Pain, 3, 37.

    Article  PubMed  CAS  Google Scholar 

  3. Ishijima, A., Kojima, H., Higuchi, H., Harada, Y., Funatsu, T., & Yanagida, T. (1996). Multiple- and single-molecule analysis of the actomyosin motor by nanometer-piconewton manipulation with a microneedle: Unitary steps and forces. Biophysical Journal, 70, 383–400.

    PubMed  CAS  Google Scholar 

  4. Svoboda, K., & Block, S. M. (1994). Force and velocity measured for single kinesin molecules. Cell, 77, 773–784.

    Article  PubMed  CAS  Google Scholar 

  5. Wang, M. D., Schnitzer, M. J., Yin, H., Landick, R., Gelles, J., & Block, S. M. (1998). Force and velocity measured for single molecules of RNA polymerase. Science, 282, 902–907.

    Article  PubMed  CAS  Google Scholar 

  6. Hua, W., Young, E. C., Fleming, M. L., & Gelles, J. (1997). Coupling of kinesin steps to ATP hydrolysis. Nature, 388, 390–393.

    Article  PubMed  CAS  Google Scholar 

  7. Schnitzer, M. J., & Block, S. M. (1997). Kinesin hydrolyses one ATP per 8-nm step. Nature, 388, 386–389.

    Article  PubMed  CAS  Google Scholar 

  8. Lu, H., Macosko, J., Habel-Rodriguez, D., Keller, R. W., Brozik, J. A., & Keller, D. J. (2004). Closing of the fingers domain generates motor forces in the HIV reverse transcriptase. Journal of Biological Chemistry, 279, 54529–54532.

    Article  PubMed  CAS  Google Scholar 

  9. Keller, D. J., & Brozik, J. A. (2005). Framework model for DNA polymerases. Biochemistry, 44, 6877–6888.

    Article  PubMed  CAS  Google Scholar 

  10. Dixit, R., Ross, J. L., Goldman, Y. E., & Holzbaur, E. L. (2008). Differential regulation of dynein and kinesin motor proteins by tau. Science, 319, 1086–1089.

    Article  PubMed  CAS  Google Scholar 

  11. Dewitt, D. A., Hurd, J. A., Fox, N., Townsend, B. E., Griffioen, K. J., Ghribi, O., et al. (2006). Peri-nuclear clustering of mitochondria is triggered during aluminum maltolate induced apoptosis. Journal of Alzheimers Disease, 9, 195–205.

    Google Scholar 

  12. Lazarov, O., Morfini, G. A., Pigino, G., Gadadhar, A., Chen, X., Robinson, J., et al. (2007). Impairments in fast axonal transport and motor neuron deficits in transgenic mice expressing familial Alzheimer’s disease-linked mutant presenilin 1. Journal of Neuroscience, 27, 7011–7020.

    Article  PubMed  CAS  Google Scholar 

  13. Ebneth, A., Godemann, R., Stamer, K., Illenberger, S., Trinczek, B., & Mandelkow, E. (1998). Overexpression of tau protein inhibits kinesin-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum: Implications for Alzheimer’s disease. Journal of Cell Biology, 143, 777–794.

    Article  PubMed  CAS  Google Scholar 

  14. Pigino, G., Morfini, G., Pelsman, A., Mattson, M. P., Brady, S. T., & Busciglio, J. (2003). Alzheimer’s presenilin 1 mutations impair kinesin-based axonal transport. Journal of Neuroscience, 23, 4499–4508.

    PubMed  CAS  Google Scholar 

  15. Kamal, A., Almenar-Queralt, A., LeBlanc, J. F., Roberts, E. A., & Goldstein, L. S. (2001). Kinesin-mediated axonal transport of a membrane compartment containing beta-secretase and presenilin-1 requires APP. Nature, 414, 643–648.

    Article  PubMed  CAS  Google Scholar 

  16. Smith, K. D., Kallhoff, V., Zheng, H., & Pautler, R. G. (2007). In vivo axonal transport rates decrease in a mouse model of Alzheimer’s disease. Neuroimage, 35, 1401–1408.

    Article  PubMed  Google Scholar 

  17. Segel, I. H. (1975). Enzyme kinetics : behavior and analysis of rapid equilibrium and steady state enzyme systems. New York: Wiley.

    Google Scholar 

  18. Perutz, M. F. (1989). Mechanisms of cooperativity and allosteric regulation in proteins. Quarterly Reviews of Biophysics, 22, 139–237.

    PubMed  CAS  Google Scholar 

  19. Perutz, M. F., & TenEyck, L. F. (1972). Stereochemistry of cooperative effects in hemoglobin. Cold Spring Harbor Symposia on Quantitative Biology, 36, 295–310.

    PubMed  CAS  Google Scholar 

  20. Ryu, W. S., Berry, R. M., & Berg, H. C. (2000). Torque-generating units of the flagellar motor of Escherichia coli have a high duty ratio. Nature, 403, 444–447.

    Article  PubMed  CAS  Google Scholar 

  21. Ashkin, A., Schuetze, K., Dziedzic, J. M., Euteneuer, U., & Schliwa, M. (1990). Force generation of organelle transport measured in vivo by an infrared laser trap. Nature, 348, 346–348.

    Article  PubMed  CAS  Google Scholar 

  22. Hirokawa, N. (1998). Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science, 279, 519–526.

    Article  PubMed  CAS  Google Scholar 

  23. Maier, B., Chen, I., Dubnau, D., & Sheetz, M. P. (2004). DNA transport into Bacillus subtilis requires proton motive force to generate large molecular forces. Nature Structural & Molecular Biology, 11, 643–649.

    Article  CAS  Google Scholar 

  24. Smith, D. E., Tans, S. J., Smith, S. B., Grimes, S., Anderson, D. L., & Bustamante, C. (2001). The bacteriophage straight phi29 portal motor can package DNA against a large internal force. Nature, 413, 748–752.

    Article  PubMed  CAS  Google Scholar 

  25. Wilcox, A. J., Choy, J., Bustamante, C., & Matouschek, A. (2005). Effect of protein structure on mitochondrial import. Proceedings of the National Academy of Sciences of the United States of America, 102, 15435–15440.

    Article  PubMed  CAS  Google Scholar 

  26. Welte, M. A., Gross, S., Postner, M., Block, S., & Wieschaus, E. (1998). Developmental regulation of vesicle transport in Drosophila embryos: Forces and kinetics. Cell, 92, 547–557.

    Article  PubMed  CAS  Google Scholar 

  27. Vershinin, M., Carter, B. C., Razafsky, D. S., King, S. J., & Gross, S. P. (2007). Multiple-motor based transport and its regulation by Tau. Proceedings of the National Academy of Sciences of the United States of America, 104, 87–92.

    Article  PubMed  CAS  Google Scholar 

  28. Friberg, H., Ferrand-Drake, M., Bengtsson, F., Halestrap, A. P., & Wieloch, T. (1998). Cyclosporin A, but not FK 506, protects mitochondria and neurons against hypoglycemic damage and implicates the mitochondrial permeability transition in cell death. Journal of Neuroscience, 18, 5151–5159.

    PubMed  CAS  Google Scholar 

  29. Bednarski, E., Ribak, C. E., & Lynch, G. (1997). Suppression of cathepsins B and L causes a proliferation of lysosomes and the formation of meganeurites in hippocampus. Journal of Neuroscience, 17, 4006–4021.

    PubMed  CAS  Google Scholar 

  30. Moreno, S., Nardacci, R., & Ceru, M. P. (1997). Regional and ultrastructural immunolocalization of copper-zinc superoxide dismutase in rat central nervous system. Journal of Histochemistry and Cytochemistry, 45, 1611–1622.

    PubMed  CAS  Google Scholar 

  31. Martin, E. J., Kim, M., Velier, J., Sapp, E., Lee, H. S., Laforet, G., et al. (1999). Analysis of Huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons. Journal of Comparative Neurology, 403, 421–430.

    Article  PubMed  CAS  Google Scholar 

  32. Stokes, G. G. (1851). On the effect of the internal friction of fluids on the motion of pendulums. Cambridge Philosophical Society Transactions, 9, 8–106.

    Google Scholar 

  33. Hill, D. B. (2003). Changes in the number of molecular motors driving vesicle transport in PC12. In his physics Ph.D. dissertation, Wake Forest University, Winston-Salem.

  34. Chisena, E. N., Wall, R. A., Macosko, J. C., & Holzwarth, G. (2007). Speckled microtubules improve tracking in motor-protein gliding assays. Physical Biology, 4, 10–15.

    Article  PubMed  CAS  Google Scholar 

  35. Cribb, J. (Retrieved Aug 4, 2008 from http://www.cs.unc.edu/Research/nano/cismm/download/spottracker/video_spot_tracker.html).

  36. Mason, T. G. (2000). Estimating the viscoelastic moduli of complex fluids using the generalized Stokes–Einstein equation. Rheologica Acta, 39, 371–378.

    Article  CAS  Google Scholar 

  37. Hill, D. B., Plaza, M. J., Bonin, K., & Holzwarth, G. (2004). Fast vesicle transport in PC12 neurites: Velocities and forces. European Biophysics Journal, 33, 623–632.

    Article  PubMed  CAS  Google Scholar 

  38. Zahn, T. R., Angleson, J. K., MacMorris, M. A., Domke, E., Hutton, J. F., Schwartz, C., et al. (2004). Dense core vesicle dynamics in Caenorhabditis elegans neurons and the role of kinesin UNC-104. Traffic, 5, 544–559.

    Article  PubMed  CAS  Google Scholar 

  39. Young, D., Glasbey, C. A., Gray, A. J., & Martin, N. J. (1998). Towards automatic cell identification in DIC microscopy. Journal of Microscopy, 192, 186–193.

    Article  PubMed  CAS  Google Scholar 

  40. Valberg, P. A., & Feldman, H. A. (1987). Magnetic particle motions within living cells measurement of cytoplasmic viscosity and motile activity. Biophysical Journal, 52, 551–561.

    Article  PubMed  CAS  Google Scholar 

  41. Holzwarth, G., Bonin, K., & Hill, D. B. (2002). Forces required of kinesin during processive transport through cytoplasm. Biophysical Journal, 82, 1784–1790.

    PubMed  CAS  Google Scholar 

  42. Breuer, A. C., Christian, C. N., Henkart, M., & Nelson, P. G. (1975). Computer analysis of organelle translocation in primary neuronal cultures and continuous cell lines. Journal of Cell Biology, 65, 562–576.

    Article  PubMed  CAS  Google Scholar 

  43. Kural, C., Kim, H., Syed, S., Goshima, G., Gelfand,. V. I., & Selvin, P. R. (2005). Kinesin and dynein move a peroxisome in vivo: A tug-of-war or coordinated movement? Science, 308, 1469–1472.

    Article  PubMed  CAS  Google Scholar 

  44. Macosko, J. C., Newbern, J. M., Rockford, J., Chisena, E. N., Brown, C. M., Holzwarth, G., & Milligan, C. E. (2008). Fewer motors per vesicle explains slowed vesicle transport in chick motoneurons after three days in vitro. Brain Research, 1211, 6–12.

    Article  PubMed  CAS  Google Scholar 

  45. Levi, V., Serpinskaya, A. S., Gratton, E., & Gelfand, V. (2006). Organelle transport along microtubules in Xenopus melanophores: Evidence for cooperation between multiple motors. Biophysical Journal, 90, 318–327.

    Article  PubMed  CAS  Google Scholar 

  46. Martinez, J. E., Vershinin, M. D., Shubeita, G. T., & Gross, S. P. (2007). On the use of in vivo cargo velocity as a biophysical marker. Biochemical and Biophysical Research Communications, 353, 835–840.

    Article  PubMed  CAS  Google Scholar 

  47. Kawaguchi, K., & Ishiwata, S. (2000). Temperature dependence of force, velocity, and processivity of single kinesin molecules. Biochemical and Biophysical Research Communications, 272, 895–899.

    Article  PubMed  CAS  Google Scholar 

  48. Meyhofer, E., & Howard, J. (1995). The force generated by a single kinesin molecule against an elastic load. Proceedings of the National Academy of Sciences of the United States of America, 92, 574–578.

    Article  PubMed  CAS  Google Scholar 

  49. Visscher, K., Schnitzer, M. J., & Block, S. M. (1999). Single kinesin molecules studied with a molecular force clamp. Nature, 400, 184–189.

    Article  PubMed  CAS  Google Scholar 

  50. Meyhoefer, E., & Howard, J. (1995). The force generated by a single kinesin molecule against an elastic load. Proceedings of the National Academy of Sciences of the United States of America, 92, 574–578.

    Article  CAS  Google Scholar 

  51. Hill, D. B., Macosko, J. C., & Holzwarth, G. M. (accepted). Motion-enhanced, differential interference contrast (MEDIC) microscopy of moving vesicles in live cells: VE-DIC updated. Journal of Microscopy.

  52. Goldstein, L. S. B. (2003). Do disorders of movement cause movement disorders and dementia? Neuron, 40, 415–425.

    Article  PubMed  CAS  Google Scholar 

  53. Weihs, D., Mason, T. G, Teitell, & M. A. (2007). Effects of cytoskeletal disruption on transport, structure, and rheology within mammalian cells. Physics of Fluids, 19, 103102-1–103102-6.

    Google Scholar 

  54. Block, S. M., Goldstein, L., & BJ, S. (1990). Bead Movement by single kinesin molecules studied with optical tweezers. Nature, 348, 348–352.

    Article  PubMed  CAS  Google Scholar 

  55. Courty, S., Luccardini, C., Bellaiche, Y., Cappello, G., & Dahan, M. (2006). Tracking individual kinesin motors in living cells using single quantum-dot imaging. Nano Letters, 6, 1491–1495.

    Article  PubMed  CAS  Google Scholar 

  56. Petrov, D. Y., Mallik, R., Shubeita, G. T., Vershinin, M., Gross, S. P., & Yu, C. C. (2007). Studying molecular motor-based cargo transport: What is real and what is noise? Biophysical Journal, 92, 2953–2963.

    Article  PubMed  CAS  Google Scholar 

  57. Hunt, A. J., Gittes, F., & Howard, J. (1994). The force exerted by a single kinesin molecule against a viscous load. Biophysical Journal, 67, 766–781.

    PubMed  CAS  Google Scholar 

  58. Brady, S. T., Lasek, R. J., & Allen, R. D. (1982). Fast axonal transport in extruded axoplasm from squid giant axon. Science, 218, 1129–1131.

    Article  PubMed  CAS  Google Scholar 

  59. Grafstein, B., & Forman, D. S. (1980). Intracellular-tranpsort in neurons. Physiological Review, 60, 1167–1283.

    CAS  Google Scholar 

  60. Kaether, C., Skehel, P., & Dotti, C. G. (2000). Axonal membrane proteins are transported in distinct carriers: A two-color video microscopy study in cultured hippocampal neurons. Molecular Biology of the Cell, 11, 1213–1224.

    PubMed  CAS  Google Scholar 

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Acknowledgments

We thank George Holzwarth for help with preparing this manuscript and Keith Bonin for helpful discussions. We gratefully acknowledge Todd Fallesen and Clayton Bauer for input at various stages. This work was supported by a start-up grant by Wake Forest University to JCM and by an NIH grant (AG-020996) to DAD.

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Authors and Affiliations

  1. Department of Physics, Wake Forest University, Winston-Salem, NC, 27109, USA

    Yuri Shtridelman, Thomas Cahyuti & Jed C. Macosko

  2. Department of Biology and Chemistry, Liberty University, Lynchburg, VA, 24502, USA

    Brigitte Townsend & David DeWitt

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  1. Yuri Shtridelman
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  2. Thomas Cahyuti
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  3. Brigitte Townsend
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  5. Jed C. Macosko
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Correspondence to Jed C. Macosko.

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Shtridelman, Y., Cahyuti, T., Townsend, B. et al. Force–Velocity Curves of Motor Proteins Cooperating In Vivo. Cell Biochem Biophys 52, 19–29 (2008). https://doi.org/10.1007/s12013-008-9021-8

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