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Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts

Nature Protocols volume 1, pages 2305–2314 (2006)Cite this article

Abstract

Extracts from Xenopus laevis eggs provide a powerful system for the study of cell division processes in vitro through biochemical reconstitution and manipulation, and microscopic analysis. We provide protocols for the preparation of metaphase-arrested extracts and in vitro assays to examine the following pathways of spindle assembly: 1) Sperm nuclei added to meiotic extracts, supporting the formation of half-spindles and bipolar spindle structures around unreplicated chromosomes; 2) sperm nuclei added to extracts that cycle through interphase and form spindles that are capable of undergoing anaphase and chromosome segregation; and 3) spindle formation around chromatin-coated beads. Finally, we describe methods to inhibit a specific protein by immunodepletion or addition of an inhibitor such as a dominant-negative construct. These techniques can be used to analyze the mitotic function of a given protein. It takes ∼1.5 h to prepare the extract, 1–3 h for spindle-assembly experiments and an additional 1–3 h if immunodepletion is performed.

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Figure 1: Schematic representation of CSF extract preparation and spindle-assembly reactions.
Figure 2: Schematic of Corex® tubes equipped with custom-made adapters: a plastic insert is sealed or placed at the bottom of the tube.
Figure 3: Examples of CSF, cycled and bead spindle reactions visualized by fluorescence microscopy, showing microtubules and DNA.

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References

  1. Murray, A.W. & Kirschner, M.W. Cyclin synthesis drives the early embryonic cell cycle. Nature 339, 275–280 (1989).

    Article  CAS  Google Scholar 

  2. Verde, F., Labbe, J.C., Doree, M. & Karsenti, E. Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature 343, 233–238 (1990).

    Article  CAS  Google Scholar 

  3. Belmont, L.D., Hyman, A.A., Sawin, K.E. & Mitchison, T.J. Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 62, 579–589 (1990).

    Article  CAS  Google Scholar 

  4. Hirano, T. & Mitchison, T.J. Cell cycle control of higher-order chromatin assembly around naked DNA in vitro. J. Cell Biol. 115, 1479–1489 (1991).

    Article  CAS  Google Scholar 

  5. Lohka, M.J. & Masui, Y. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220, 719–721 (1983).

    Article  CAS  Google Scholar 

  6. Desai, A., Deacon, H.W., Walczak, C.E. & Mitchison, T.J. A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts. Proc. Natl. Acad. Sci. USA 94, 12378–12383 (1997).

    Article  CAS  Google Scholar 

  7. Sawin, K.E. & Mitchison, T.J. Mitotic spindle assembly by two different pathways in vitro. J. Cell Biol. 112, 925–940 (1991).

    Article  CAS  Google Scholar 

  8. Lohka, M.J. & Maller, J.L. Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J. Cell Biol. 101, 518–523 (1985).

    Article  CAS  Google Scholar 

  9. Desai, A., Maddox, P.S., Mitchison, T.J. & Salmon, E.D. Anaphase A chromosome movement and poleward spindle microtubule flux occur at similar rates in Xenopus extract spindles. J. Cell Biol. 141, 703–713 (1998).

    Article  CAS  Google Scholar 

  10. Murray, A.W., Desai, A.B. & Salmon, E.D. Real time observation of anaphase in vitro. Proc. Natl. Acad. Sci. USA 93, 12327–12332 (1996).

    Article  CAS  Google Scholar 

  11. Shamu, C.E. & Murray, A.W. Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase. J. Cell Biol. 117, 921–934 (1992).

    Article  CAS  Google Scholar 

  12. Schmidt, A., Rauh, N.R., Nigg, E.A. & Mayer, T.U. Cytostatic factor: an activity that puts the cell cycle on hold. J. Cell Sci. 119, 1213–1218 (2006).

    Article  CAS  Google Scholar 

  13. Heald, R. et al. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 382, 420–425 (1996).

    Article  CAS  Google Scholar 

  14. Budde, P.P. & Heald, R. Centrosomes and kinetochores, who needs 'Em? The role of noncentromeric chromatin in spindle assembly. Curr. Top. Dev. Biol. 56, 85–113 (2003).

    Article  CAS  Google Scholar 

  15. Maiato, H., Rieder, C.L. & Khodjakov, A. Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis. J. Cell Biol. 167, 831–840 (2004).

    Article  CAS  Google Scholar 

  16. Heald, R., Tournebize, R., Habermann, A., Karsenti, E. & Hyman, A. Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization. J. Cell Biol. 138, 615–628 (1997).

    Article  CAS  Google Scholar 

  17. Murray, A. in Methods in Cell Biology (eds. Kay, B.K. & Peng, H.B.) 581–605 (Academic Press, San Diego, 1991).

    Google Scholar 

  18. Vernos, I. et al. Xklp1, a chromosomal Xenopus kinesin-like protein essential for spindle organization and chromosome positioning. Cell 81, 117–127 (1995).

    Article  CAS  Google Scholar 

  19. 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 

  20. Wignall, S.M. et al. Identification of a novel protein regulating microtubule stability through a chemical approach. Chem. Biol. 11, 135–146 (2004).

    Article  CAS  Google Scholar 

  21. Wu, M. & Gerhart, J. in Methods in Cell Biology (eds. Kay, B.K. & Peng, H.B.) 3–18 (Academic Press, San Diego, 1991).

    Google Scholar 

  22. Hyman, A. et al. in Methods in Enzymology (ed. Vallee, R.B.) 478–485 (Academic Press, San Diego, 1991).

    Google Scholar 

  23. Morin, N., Abrieu, A., Lorca, T., Martin, F. & Doree, M. The proteolysis-dependent metaphase to anaphase transition: calcium/calmodulin-dependent protein kinase II mediates onset of anaphase in extracts prepared from unfertilized Xenopus eggs. EMBO J. 13, 4343–4352 (1994).

    Article  CAS  Google Scholar 

  24. Gaglio, T. et al. Opposing motor activities are required for the organization of the mammalian mitotic spindle pole. J. Cell Biol. 135, 399–414 (1996).

    Article  CAS  Google Scholar 

  25. Merdes, A., Ramyar, K., Vechio, J.D. & Cleveland, D.W. A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly. Cell 87, 447–458 (1996).

    Article  CAS  Google Scholar 

  26. Sawin, K.E., LeGuellec, K., Philippe, M. & Mitchison, T.J. Mitotic spindle organization by a plus-end-directed microtubule motor. Nature 359, 540–543 (1992).

    Article  CAS  Google Scholar 

  27. 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 

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  1. University of California, Berkeley, 315 Life Sciences Addition, Berkeley, 94720-3200, CA, USA

    Eva Hannak & Rebecca Heald

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Correspondence to Rebecca Heald.

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Hannak, E., Heald, R. Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts. Nat Protoc 1, 2305–2314 (2006). https://doi.org/10.1038/nprot.2006.396

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