Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Microneedle-based analysis of the micromechanics of the metaphase spindle assembled in Xenopus laevis egg extracts

Abstract

To explain how micrometer-sized cellular structures generate and respond to forces, we need to characterize their micromechanical properties. Here we provide a protocol to build and use a dual force-calibrated microneedle-based setup to quantitatively analyze the micromechanics of a metaphase spindle assembled in Xenopus laevis egg extracts. This cell-free extract system allows for controlled biochemical perturbations of spindle components. We describe how the microneedles are prepared and how they can be used to apply and measure forces. A multimode imaging system allows the tracking of microtubules, chromosomes and needle tips. This setup can be used to analyze the viscoelastic properties of the spindle on timescales ranging from minutes to sub-seconds. A typical experiment, along with data analysis, is also detailed. We anticipate that our protocol can be readily extended to analyze the micromechanics of other cellular structures assembled in cell-free extracts. The entire procedure can take 3–4 d.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Experimental setup for analysis of metaphase spindle micromechanics.
Figure 2: Calibration of the reference needle.
Figure 3: Fabrication of the microneedles.
Figure 4: Calibration of the microneedles.
Figure 5: An example of an experiment and analysis.

Similar content being viewed by others

References

  1. Brenner, M.D., Zhou, R. & Ha, T. Forcing a connection: impacts of single-molecule force spectroscopy on in vivo tension sensing. Biopolymers 95, 332–344 (2011).

    Article  CAS  Google Scholar 

  2. Neuman, K.C. & Nagy, A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat. Methods 5, 491–505 (2008).

    Article  CAS  Google Scholar 

  3. Veigel, C. & Schmidt, C.F. Moving into the cell: single-molecule studies of molecular motors in complex environments. Nat. Rev. Mol. Cell Biol. 12, 163–176 (2011).

    Article  CAS  Google Scholar 

  4. Inoue, S. & Salmon, E.D. Force generation by microtubule assembly/disassembly in mitosis and related movements. Mol. Biol. Cell 6, 1619–1640 (1995).

    Article  CAS  Google Scholar 

  5. Waters, J.C., Skibbens, R.V. & Salmon, E.D. Oscillating mitotic newt lung cell kinetochores are, on average, under tension and rarely push. J. Cell. Sci. 109 (Part 12): 2823–2831 (1996).

    Article  CAS  Google Scholar 

  6. Nicklas, R.B. Chromosome velocity during mitosis as a function of chromosome size and position. J. Cell. Biol. 25 (suppl): 119–135 (1965).

    Article  Google Scholar 

  7. Grill, S.W., Howard, J., Schaffer, E., Stelzer, E.H. & Hyman, A.A. The distribution of active force generators controls mitotic spindle position. Science 301, 518–521 (2003).

    Article  CAS  Google Scholar 

  8. Gardel, M.L., Kasza, K.E., Brangwynne, C.P., Liu, J. & Weitz, D.A. Mechanical response of cytoskeletal networks. Methods Cell Biol. 89, 487–519 (2008).

    Article  CAS  Google Scholar 

  9. Lin, Y.-C., Koenderink, G.H., MacKintosh, F.C. & Weitz, D.A. Viscoelastic properties of microtubule networks. Macromolecules 40, 7714–7720 (2007).

    Article  CAS  Google Scholar 

  10. Murray, A.W. Cell cycle extracts. Methods Cell Biol. 36, 581–605 (1991).

    Article  CAS  Google Scholar 

  11. Hannak, E. & Heald, R. Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts. Nat. Protoc. 1, 2305–2314 (2006).

    Article  CAS  Google Scholar 

  12. Kapoor, T.M. & Mitchison, T.J. Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix. J. Cell Biol. 154, 1125–1133 (2001).

    Article  CAS  Google Scholar 

  13. Miyamoto, D.T., Perlman, Z.E., Burbank, K.S., Groen, A.C. & Mitchison, T.J. The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles. J. Cell Biol. 167, 813–818 (2004).

    Article  CAS  Google Scholar 

  14. Walczak, C.E., Mitchison, T.J. & Desai, A. XKCM1: a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly. Cell 84, 37–47 (1996).

    Article  CAS  Google Scholar 

  15. Houghtaling, B.R., Yang, G., Matov, A., Danuser, G. & Kapoor, T.M. Op18 reveals the contribution of nonkinetochore microtubules to the dynamic organization of the vertebrate meiotic spindle. Proc. Natl. Acad. Sci. USA 106, 15338–15343 (2009).

    Article  CAS  Google Scholar 

  16. Wittmann, T. & Hyman, T. Recombinant p50/dynamitin as a tool to examine the role of dynactin in intracellular processes. Methods Cell Biol. 61, 137–143 (1999).

    Article  CAS  Google Scholar 

  17. Gatlin, J.C., Matov, A., Danuser, G., Mitchison, T.J. & Salmon, E.D. Directly probing the mechanical properties of the spindle and its matrix. J. Cell Biol. 188, 481–489 (2010).

    Article  CAS  Google Scholar 

  18. Shimamoto, Y., Maeda, Y.T., Ishiwata, S., Libchaber, A.J. & Kapoor, T.M. Insights into the micromechanical properties of the metaphase spindle. Cell 145, 1062–1074 (2011).

    Article  CAS  Google Scholar 

  19. Nicklas, R.B. How cells get the right chromosomes. Science 275, 632–637 (1997).

    Article  CAS  Google Scholar 

  20. Shimamoto, Y., Suzuki, M., Mikhailenko, S.V., Yasuda, K. & Ishiwata, S. Inter-sarcomere coordination in muscle revealed through individual sarcomere response to quick stretch. Proc. Natl. Acad. Sci. USA 106, 11954–11959 (2009).

    Article  CAS  Google Scholar 

  21. Howard, J. & Hudspeth, A.J. Mechanical relaxation of the hair bundle mediates adaptation in mechanoelectrical transduction by the bullfrog's saccular hair cell. Proc. Natl. Acad. Sci. USA 84, 3064–3068 (1987).

    Article  CAS  Google Scholar 

  22. Okuno, M. & Hiramoto, Y. Direct measurements of the stiffness of echinoderm sperm flagella. J. Exp. Biol. 79, 235–243 (1979).

    Article  CAS  Google Scholar 

  23. Svoboda, K. & Block, S.M. Biological applications of optical forces. Annu. Rev. Biophys. Biomol. Struct. 23, 247–285 (1994).

    Article  CAS  Google Scholar 

  24. Tanase, M., Biais, N. & Sheetz, M. Magnetic tweezers in cell biology. Methods Cell Biol. 83, 473–493 (2007).

    Article  CAS  Google Scholar 

  25. Fisher, T.E., Marszalek, P.E. & Fernandez, J.M. Stretching single molecules into novel conformations using the atomic force microscope. Nat. Struct. Biol. 7, 719–724 (2000).

    Article  CAS  Google Scholar 

  26. Janmey, P.A., Georges, P.C. & Hvidt, S. Basic rheology for biologists. Methods Cell Biol. 83, 3–27 (2007).

    CAS  PubMed  Google Scholar 

  27. Theriot, J.A., Rosenblatt, J., Portnoy, D.A., Goldschmidt-Clermont, P.J. & Mitchison, T.J. Involvement of profilin in the actin-based motility of L. monocytogenes in cells and in cell-free extracts. Cell 76, 505–517 (1994).

    Article  CAS  Google Scholar 

  28. Gaglio, T., Saredi, A. & Compton, D.A. NuMA is required for the organization of microtubules into aster-like mitotic arrays. J. Cell Biol. 131, 693–708 (1995).

    Article  CAS  Google Scholar 

  29. Lee, K., Gallop, J.L., Rambani, K. & Kirschner, M.W. Self-assembly of filopodia-like structures on supported lipid bilayers. Science 329, 1341–1345 (2010).

    Article  CAS  Google Scholar 

  30. Stearns, T. & Kirschner, M. In vitro reconstitution of centrosome assembly and function: the central role of gamma-tubulin. Cell 76, 623–637 (1994).

    Article  CAS  Google Scholar 

  31. Levy, D.L. & Heald, R. Nuclear size is regulated by importin α and Ntf2 in Xenopus. Cell 143, 288–298 (2010).

    Article  CAS  Google Scholar 

  32. Hyman, A. et al. Preparation of modified tubulins. Methods Enzymol. 196, 478–485 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

T.M.K. acknowledges support from NIH/NIGMS (GM065933). Y.S. was a recipient of the Uehara memorial foundation postdoctoral fellowship and is supported by the JSPS postdoctoral fellowship for research abroad.

Author information

Authors and Affiliations

Authors

Contributions

Y.S. developed the protocol. Y.S. and T.M.K. prepared the manuscript.

Corresponding author

Correspondence to Tarun M Kapoor.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shimamoto, Y., Kapoor, T. Microneedle-based analysis of the micromechanics of the metaphase spindle assembled in Xenopus laevis egg extracts. Nat Protoc 7, 959–969 (2012). https://doi.org/10.1038/nprot.2012.033

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.033

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing