Microtubule Dynamics pp 147-165

Part of the Methods in Molecular Biology book series (MIMB, volume 777)

Force Generation by Dynamic Microtubules In Vitro

  • Svenja-Marei Kalisch
  • Liedewij Laan
  • Marileen Dogterom


Biopolymers are essential for cellular organization. They bridge the cell interior, forming a framework that is used as a reference for different cellular organelles. This framework, called the cytoskeleton, is not static but constantly reorganizes. The dynamics of the cytoskeleton allows the cell to rearrange its interior for various processes, such as cell division. This dynamic reorganization relies at least partly on forces that arise from the assembly and disassembly of cytoskeletal biopolymers. In many cases, these forces are generated when biopolymers interact with the cell boundary. This chapter focuses on force generation by and regulation of microtubules (MTs) that interact with growth-opposing barriers. We describe three in vitro assays that can be used to mimic MT interactions with the cell boundary. The essential components in each of our minimal systems are (functionalized) microfabricated barriers against which we grow MTs under different conditions. We describe in detail the different methods and assays necessary to realize these in vitro experiments.

Key words

Cytoskeleton Microtubules Microfabrication Force Glass barriers Gold barriers SU-8 barriers Optical tweezers Force feedback 


  1. 1.
    Inoue, S., and Salmon, E. D. (1995) Force Generation by Microtubule Assembly Disassembly in Mitosis and Related Movements. Mol Biol Cell 6, 1619–1640PubMedGoogle Scholar
  2. 2.
    Adames, N. R., and Cooper, J. A. (2000) Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae. J Cell Biol 149, 863–874PubMedCrossRefGoogle Scholar
  3. 3.
    Dogterom, M., Kerssemakers, J. W., Romet-Lemonne, G., and Janson, M. E. (2005) Force generation by dynamic microtubules. Curr Opin Cell Biol 17, 67–74PubMedCrossRefGoogle Scholar
  4. 4.
    Tolic-Norrelykke, I. M., Sacconi, L., Stringari, C., Raabe, I., and Pavone, F. S. (2005) Nuclear and division-plane positioning revealed by optical micromanipulation. Current Biology 15, 1212–1216PubMedCrossRefGoogle Scholar
  5. 5.
    Tran, P., Marsh, L., Doye, V., Inoue, S., and Chang, F. (2001) A mechanism for nuclear positioning in fission yeast based on microtubule pushing. J. Cell Biol. 153, 397–411PubMedCrossRefGoogle Scholar
  6. 6.
    Mitchison, J., and Kirschner, M. (1984) Dynamic instbility of microtubule growth. Nature 312, 237–242PubMedCrossRefGoogle Scholar
  7. 7.
    Mitchison, J., and Kirschner, M. (1984) Microtubule assembly nucleated by isolated centrosomes. Nature 312, 232–237PubMedCrossRefGoogle Scholar
  8. 8.
    Desai, A., and Mitchison, T. J. (1997) Microtubule polymerization dynamics. Annu Rev Cell Dev Biol 13, 83–117PubMedCrossRefGoogle Scholar
  9. 9.
    Fygenson, D. K., Braun, E., and Libchaber, A. (1994) Phase-Diagram of Microtubules. Physical Review E 50, 1579–1588CrossRefGoogle Scholar
  10. 10.
    Gadde, S., and Heald, R. (2004) Mechanisms and molecules of the mitotic spindle. Current Biology 14, R797–R805PubMedCrossRefGoogle Scholar
  11. 11.
    Bieling, P., Kandels-Lewis, S., Telley, I. A., van Dijk, J., Janke, C., and Surrey, T. (2008) CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites. Journal of Cell Biology 183, 1223–1233PubMedCrossRefGoogle Scholar
  12. 12.
    Bieling, P., Laan, L., Schek, H., Munteanu, E. L., Sandblad, L., Dogterom, M., Brunner, D., and Surrey, T. (2007) Reconstitution of a microtubule plus-end tracking system in vitro. Nature 450, 1100–1105PubMedCrossRefGoogle Scholar
  13. 13.
    Brouhard, G. J., Stear, J. H., Noetzel, T. L., Al-Bassam, J., Kinoshita, K., Harrison, S. C., Howard, J., and Hyman, A. A. (2008) XMAP215 is a processive microtubule polymerase. Cell 132, 79–88PubMedCrossRefGoogle Scholar
  14. 14.
    Dixit, R., Barnett, B., Lazarus, J. E., Tokito, M., Goldman, Y. E., and Holzbaur, E. L. F. (2009) Microtubule plus-end tracking by CLIP-170 requires EB1. Proc Natl Acad Sci USA 106, 492–497PubMedCrossRefGoogle Scholar
  15. 15.
    Honnappa, S., Gouveia, S. M., Weisbrich, A., Damberger, F. F., Bhavesh, N. S., Jawhari, H., Grigoriev, I., van Rijssel, F. J., Buey, R. M., Lawera, A., Jelesarov, I., Winkler, F. K., Wuthrich, K., Akhmanova, A., and Steinmetz, M. O. (2009) An EB1-binding motif acts as a microtubule tip localization signal. Cell 138, 366–76PubMedCrossRefGoogle Scholar
  16. 16.
    Kinoshita, K., Arnal, I., Desai, A., Drechsel, D. N., and Hyman, A. A. (2001) Reconstitution of physiological microtubule dynamics using purified components. Science 294, 1340–1343PubMedCrossRefGoogle Scholar
  17. 17.
    Kinoshita, K., Habermann, B., and Hyman, A. A. (2002) XMAP215: a key component of the dynamic microtubule cytoskeleton. Trends in Cell Biology 12, 267–273PubMedCrossRefGoogle Scholar
  18. 18.
    Komarova, Y., De Groot, C. O., Grogoriev, I., Montenegro Gouveia, S., Munteanu, E. L., Schober, J. M., Honnappa, S., Buey, R. M., Hoogenraad, C. C., Dogterom, M., Borisy, G., Steinmetz, M., and Akhmanova, A. (2009) Mammalian end binding proteins control persistent microtubule growth. J Cell Biol 184, 691–706PubMedCrossRefGoogle Scholar
  19. 19.
    Dogterom, M., and Yurke, B. (1997) Measurement of the force-velocity relation for growing microtubules. Science 278, 856–860CrossRefGoogle Scholar
  20. 20.
    Fygenson, D. K., Marko, J. F., and Libchaber, A. (1997) Mechanics of microtubule-based membrane extension. Phys. Rev. Lett. 79, 4497–4500Google Scholar
  21. 21.
    Janson, M. E. (2002) Force Generation by Growing Microtubules. PhD thesis Google Scholar
  22. 22.
    Janson, M. E., and Dogterom, M. (2004) A bending mode analysis for growing microtubules: Evidence for a velocity-dependent rigidity. Biophysical Journal 87, 2723–2736PubMedCrossRefGoogle Scholar
  23. 23.
    Janson, M. E., Dood, M. E. d., and Dogterom, M. (2003) Dynamic instability of microtubules is regulated by force. The Journal of Cell Biology 161, 1029–1034Google Scholar
  24. 24.
    Kerssemakers, J. W. J., Janson, M. E., Van der Horst, A., and Dogterom, M. (2003) Optical trap setup for measuring microtubule pushing forces. Applied Physics Letters 83, 4441–4443CrossRefGoogle Scholar
  25. 25.
    Kerssemakers, J. W. J., Munteanu, E. L., Laan, L., Noetzel, T. L., Janson, M. E., and Dogterom, M. (2006) Assembly dynamics of microtubules at molecular resolution. Nature 442, 709–712PubMedCrossRefGoogle Scholar
  26. 26.
    Finer, J. T., Simmons, R. M., and Spudich, J. A. (1994) Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature 368, 113–119PubMedCrossRefGoogle Scholar
  27. 27.
    Simmons, R. M., Finer, J. T., Chu, S., and Spudich, J. A. (1996) Quantitative measurements of force and displacement using an optical trap. Biophys J 70, 1813–1822PubMedCrossRefGoogle Scholar
  28. 28.
    Visscher, K., Schnitzer, M. J., and Block, S. M. (1999) Single kinesin molecules studied with a molecular force clamp. Nature 400, 184–189PubMedCrossRefGoogle Scholar
  29. 29.
    Schek, H. T., Gardner, M. K., Cheng, J., Odde, D. J., and Hunt, A. J. (2007) Microtubule assembly dynamics at the nanoscale. Current Biology 17, 1445–1455PubMedCrossRefGoogle Scholar
  30. 30.
    Moudjou, M., and Bornens, M. (1994) Isolation of Centrosomes From Cultured Animal Cells. Cell Biology: A Laboratory Handbook, ed. J.E.Celis Academic Press, New York 595–604Google Scholar
  31. 31.
    Gibbons, I. R., and Frank, E. (1979) A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella. J. Biol. Chem. 254, 187–196PubMedGoogle Scholar
  32. 32.
    Tselutin, K., Seigneurin, F., and Blesbois, E. (1999) Comparison of cryoprotectants and methods of cryopreservation of fowl spermatozoa. Poult. Sci. 78, 586–590PubMedGoogle Scholar
  33. 33.
    Reck-Peterson, S. L., Yildiz, A., Carter, A. P., Gennerich, A., Zhang, N., and Vale, R. D. (2006) Single-molecule analysis of dynein processivity and stepping behavior. Cell 126, 335–348PubMedCrossRefGoogle Scholar
  34. 34.
    Howard, J., and Hyman, A. A. (2007) Microtubule polymerases and depolymerases. Curr Opin Cell Biol 19, 31–35PubMedCrossRefGoogle Scholar
  35. 35.
    The content of sections 1 to 3 as well as the one of Fig. 1–5 is based on Laan, L., and Dogterom, M. (2010) In vitro assays to study force generation at dynamic microtubule ends. Methods in Cell Biology in press, as well as on Laan, L. (2009) Force generation at microtubule ends: An in vitro approach to cortical interactions. PhD thesis Google Scholar
  36. 36.
    Laan, L., Husson, J., Van Duijn, M., Vale, R. D., Reck-Peterson, S. L., and Dogterom, M. (2009) Cortex-attached dynein regulates microtubule dynamics and pulls on shrinking microtubule ends in vitro. submitted Google Scholar
  37. 37.
    Schek, H. T., and Hunt, A. J. (2005) Micropatterned structures for studying the mechanics of biological polymers. Biomed. Microdevices 7, 41–46PubMedCrossRefGoogle Scholar
  38. 38.
    Dogterom, M., Felix, M.A., Guet, C.C. and Leibler, S. (1996) Influence of M-phase chromatin on the anisotropy of microtubule asters. J Cell Biol, 133, 125–140PubMedCrossRefGoogle Scholar
  39. 39.
    Romet-Lemonne, G., VanDuijn, M., and Dogterom, M. (2005) Three-dimensional control of protein patterning in microfabricated devices. Nano Letters 5, 2350–2354PubMedCrossRefGoogle Scholar
  40. 40.
    Gittes, F., and Schmidt, C. F. (1998) Signals and noise in micromechanical measurements. Methods in Cell Biology, 55, 129–156PubMedCrossRefGoogle Scholar
  41. 41.
    Ashkin, A. (1970) Acceleration and trapping of particles by radiation pressure. Physical Review Letters 24, 156–159CrossRefGoogle Scholar
  42. 42.
    Svoboda, K., and Block, S. M. (1994) Force and Velocity Measured for Single Kinesin Molecules. Cell 77, 773–784PubMedCrossRefGoogle Scholar
  43. 43.
    Laan, L., Husson, J., Munteanu, E.L., Kerssemakers, J.W.J. and Dogterom, M. (2008) Force-generation and dynamic instability of microtubule bundles. Proc Natl Acad Sci USA 105, 8920–8925PubMedCrossRefGoogle Scholar
  44. 44.
    Visscher, K., Gross, S. P., and Block, S. M. (1996) Construction of Multiple-Beam Optical Traps with Nanometer-Resolution Position Sensing. IEEE Journal of Selected Topics in Quantum Electronics 2, 1066–1076Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Svenja-Marei Kalisch
    • 1
  • Liedewij Laan
    • 1
    • 2
  • Marileen Dogterom
    • 1
  1. 1.FOM Institute for Atomic and Molecular Physics (AMOLF)AmsterdamThe Netherlands
  2. 2.Department of Molecular and Cellular BiologyHarvard UniversityCambridgeUSA

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