Search tips
Search criteria 


Logo of jcellbiolHomeEditorsContactInstructions for Authors
J Cell Biol. 1991 March 1; 112(5): 925–940.
PMCID: PMC2288871

Mitotic spindle assembly by two different pathways in vitro


We have used Xenopus egg extracts to study spindle morphogenesis in a cell-free system and have identified two pathways of spindle assembly in vitro using methods of fluorescent analogue cytochemistry. When demembranated sperm nuclei are added to egg extracts arrested in a mitotic state, individual nuclei direct the assembly of polarized microtubule arrays, which we term half-spindles; half-spindles then fuse pairwise to form bipolar spindles. In contrast, when sperm nuclei are added to extracts that are induced to enter interphase and arrested in the following mitosis, a single sperm nucleus can direct the assembly of a complete spindle. We find that microtubule arrays in vitro are strongly biased towards chromatin, but this does not depend on specific kinetochore-microtubule interactions. Indeed, although we have identified morphological and probably functional kinetochores in spindles assembled in vitro, kinetochores appear not to play an obligate role in the establishment of stable, bipolar microtubule arrays in either assembly pathway. Features of the two pathways suggest that spindle assembly involves a hierarchy of selective microtubule stabilization, involving both chromatin-microtubule interactions and antiparallel microtubule-microtubule interactions, and that fundamental molecular interactions are probably the same in both pathways. This in vitro reconstitution system should be useful for identifying the molecules regulating the generation of asymmetric microtubule arrays and for understanding spindle morphogenesis in general.

Full Text

The Full Text of this article is available as a PDF (5.2M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bajer AS. Functional autonomy of monopolar spindle and evidence for oscillatory movement in mitosis. J Cell Biol. 1982 Apr;93(1):33–48. [PMC free article] [PubMed]
  • Belmont LD, Hyman AA, Sawin KE, Mitchison TJ. Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell. 1990 Aug 10;62(3):579–589. [PubMed]
  • Blow JJ, Watson JV. Nuclei act as independent and integrated units of replication in a Xenopus cell-free DNA replication system. EMBO J. 1987 Jul;6(7):1997–2002. [PubMed]
  • Brenner S, Pepper D, Berns MW, Tan E, Brinkley BR. Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients. J Cell Biol. 1981 Oct;91(1):95–102. [PMC free article] [PubMed]
  • BRIGGS R, GREEN EU, KING TJ. An investigation of the capacity for cleavage and differentiation in Rana pipiens eggs lacking "functional" chromosomes. J Exp Zool. 1951 Apr;116(3):455–499. [PubMed]
  • Brinkley BR, Zinkowski RP, Mollon WL, Davis FM, Pisegna MA, Pershouse M, Rao PN. Movement and segregation of kinetochores experimentally detached from mammalian chromosomes. Nature. 1988 Nov 17;336(6196):251–254. [PubMed]
  • Church K, Nicklas RB, Lin HP. Micromanipulated bivalents can trigger mini-spindle formation in Drosophila melanogaster spermatocyte cytoplasm. J Cell Biol. 1986 Dec;103(6 Pt 2):2765–2773. [PMC free article] [PubMed]
  • Dinsmore JH, Sloboda RD. Calcium and calmodulin-dependent phosphorylation of a 62 kd protein induces microtubule depolymerization in sea urchin mitotic apparatuses. Cell. 1988 Jun 3;53(5):769–780. [PubMed]
  • Enos AP, Morris NR. Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans. Cell. 1990 Mar 23;60(6):1019–1027. [PubMed]
  • Evans L, Mitchison T, Kirschner M. Influence of the centrosome on the structure of nucleated microtubules. J Cell Biol. 1985 Apr;100(4):1185–1191. [PMC free article] [PubMed]
  • Gard DL, Kirschner MW. Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs. J Cell Biol. 1987 Nov;105(5):2191–2201. [PMC free article] [PubMed]
  • Gard DL, Hafezi S, Zhang T, Doxsey SJ. Centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle. J Cell Biol. 1990 Jun;110(6):2033–2042. [PMC free article] [PubMed]
  • Gorbsky GJ, Sammak PJ, Borisy GG. Microtubule dynamics and chromosome motion visualized in living anaphase cells. J Cell Biol. 1988 Apr;106(4):1185–1192. [PMC free article] [PubMed]
  • Gurdon JB. Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal. J Embryol Exp Morphol. 1976 Dec;36(3):523–540. [PubMed]
  • Hutchison CJ, Cox R, Ford CC. The control of DNA replication in a cell-free extract that recapitulates a basic cell cycle in vitro. Development. 1988 Jul;103(3):553–566. [PubMed]
  • Ikegami S, Taguchi T, Ohashi M, Oguro M, Nagano H, Mano Y. Aphidicolin prevents mitotic cell division by interfering with the activity of DNA polymerase-alpha. Nature. 1978 Oct 5;275(5679):458–460. [PubMed]
  • Karsenti E, Newport J, Hubble R, Kirschner M. Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into Xenopus eggs. J Cell Biol. 1984 May;98(5):1730–1745. [PMC free article] [PubMed]
  • Karsenti E, Newport J, Kirschner M. Respective roles of centrosomes and chromatin in the conversion of microtubule arrays from interphase to metaphase. J Cell Biol. 1984 Jul;99(1 Pt 2):47s–54s. [PMC free article] [PubMed]
  • Kirschner M, Mitchison T. Beyond self-assembly: from microtubules to morphogenesis. Cell. 1986 May 9;45(3):329–342. [PubMed]
  • Kristofferson D, Mitchison T, Kirschner M. Direct observation of steady-state microtubule dynamics. J Cell Biol. 1986 Mar;102(3):1007–1019. [PMC free article] [PubMed]
  • Lohka MJ, Masui Y. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science. 1983 May 13;220(4598):719–721. [PubMed]
  • Lohka MJ, Masui Y. The germinal vesicle material required for sperm pronuclear formation is located in the soluble fraction of egg cytoplasm. Exp Cell Res. 1983 Oct 15;148(2):481–491. [PubMed]
  • Lohka MJ, Maller JL. Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J Cell Biol. 1985 Aug;101(2):518–523. [PMC free article] [PubMed]
  • Lohka MJ, Hayes MK, Maller JL. Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. Proc Natl Acad Sci U S A. 1988 May;85(9):3009–3013. [PubMed]
  • Masuda H, McDonald KL, Cande WZ. The mechanism of anaphase spindle elongation: uncoupling of tubulin incorporation and microtubule sliding during in vitro spindle reactivation. J Cell Biol. 1988 Aug;107(2):623–633. [PMC free article] [PubMed]
  • Masui Y, Markert CL. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J Exp Zool. 1971 Jun;177(2):129–145. [PubMed]
  • Mazia D. Centrosomes and mitotic poles. Exp Cell Res. 1984 Jul;153(1):1–15. [PubMed]
  • Mazia D. The chromosome cycle and the centrosome cycle in the mitotic cycle. Int Rev Cytol. 1987;100:49–92. [PubMed]
  • Mazia D, Paweletz N, Sluder G, Finze EM. Cooperation of kinetochores and pole in the establishment of monopolar mitotic apparatus. Proc Natl Acad Sci U S A. 1981 Jan;78(1):377–381. [PubMed]
  • McIntosh JR, Koonce MP. Mitosis. Science. 1989 Nov 3;246(4930):622–628. [PubMed]
  • McIntosh JR, McDonald KL, Edwards MK, Ross BM. Three-dimensional structure of the central mitotic spindle of Diatoma vulgare. J Cell Biol. 1979 Nov;83(2 Pt 1):428–442. [PMC free article] [PubMed]
  • Meluh PB, Rose MD. KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell. 1990 Mar 23;60(6):1029–1041. [PubMed]
  • Mitchison TJ. Microtubule dynamics and kinetochore function in mitosis. Annu Rev Cell Biol. 1988;4:527–549. [PubMed]
  • Mitchison T, Kirschner M. Dynamic instability of microtubule growth. Nature. 1984 Nov 15;312(5991):237–242. [PubMed]
  • Mitchison TJ, Kirschner MW. Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation. J Cell Biol. 1985 Sep;101(3):766–777. [PMC free article] [PubMed]
  • Mitchison T, Evans L, Schulze E, Kirschner M. Sites of microtubule assembly and disassembly in the mitotic spindle. Cell. 1986 May 23;45(4):515–527. [PubMed]
  • Murray AW, Kirschner MW. Cyclin synthesis drives the early embryonic cell cycle. Nature. 1989 May 25;339(6222):275–280. [PubMed]
  • Murray AW, Kirschner MW. Dominoes and clocks: the union of two views of the cell cycle. Science. 1989 Nov 3;246(4930):614–621. [PubMed]
  • Murray AW, Solomon MJ, Kirschner MW. The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature. 1989 May 25;339(6222):280–286. [PubMed]
  • Nagano H, Hirai S, Okano K, Ikegami S. Achromosomal cleavage of fertilized starfish eggs in the presence of aphidicolin. Dev Biol. 1981 Jul 30;85(2):409–415. [PubMed]
  • Newport J, Spann T. Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes. Cell. 1987 Jan 30;48(2):219–230. [PubMed]
  • Nicklas RB. Mitosis. Adv Cell Biol. 1971;2:225–297. [PubMed]
  • Nicklas RB. The motor for poleward chromosome movement in anaphase is in or near the kinetochore. J Cell Biol. 1989 Nov;109(5):2245–2255. [PMC free article] [PubMed]
  • Nicklas RB, Gordon GW. The total length of spindle microtubules depends on the number of chromosomes present. J Cell Biol. 1985 Jan;100(1):1–7. [PMC free article] [PubMed]
  • Picard A, Harricane MC, Labbe JC, Doree M. Germinal vesicle components are not required for the cell-cycle oscillator of the early starfish embryo. Dev Biol. 1988 Jul;128(1):121–128. [PubMed]
  • Raff JW, Glover DM. Centrosomes, and not nuclei, initiate pole cell formation in Drosophila embryos. Cell. 1989 May 19;57(4):611–619. [PubMed]
  • RAPPAPORT R. Experiments concerning the cleavage stimulus in sand dollar eggs. J Exp Zool. 1961 Oct;148:81–89. [PubMed]
  • Rieder CL. The formation, structure, and composition of the mammalian kinetochore and kinetochore fiber. Int Rev Cytol. 1982;79:1–58. [PubMed]
  • Rieder CL, Davison EA, Jensen LC, Cassimeris L, Salmon ED. Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half-spindle. J Cell Biol. 1986 Aug;103(2):581–591. [PMC free article] [PubMed]
  • Sagata N, Watanabe N, Vande Woude GF, Ikawa Y. The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. Nature. 1989 Nov 30;342(6249):512–518. [PubMed]
  • Salmon ED, Segall RR. Calcium-labile mitotic spindles isolated from sea urchin eggs (Lytechinus variegatus). J Cell Biol. 1980 Aug;86(2):355–365. [PMC free article] [PubMed]
  • Salmon ED, Leslie RJ, Saxton WM, Karow ML, McIntosh JR. Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching. J Cell Biol. 1984 Dec;99(6):2165–2174. [PMC free article] [PubMed]
  • Sawin KE, Mitchison TJ. Poleward microtubule flux mitotic spindles assembled in vitro. J Cell Biol. 1991 Mar;112(5):941–954. [PMC free article] [PubMed]
  • Saxton WM, McIntosh JR. Interzone microtubule behavior in late anaphase and telophase spindles. J Cell Biol. 1987 Aug;105(2):875–886. [PMC free article] [PubMed]
  • Saxton WM, Stemple DL, Leslie RJ, Salmon ED, Zavortink M, McIntosh JR. Tubulin dynamics in cultured mammalian cells. J Cell Biol. 1984 Dec;99(6):2175–2186. [PMC free article] [PubMed]
  • Schulze E, Kirschner M. Microtubule dynamics in interphase cells. J Cell Biol. 1986 Mar;102(3):1020–1031. [PMC free article] [PubMed]
  • Shpetner HS, Vallee RB. Identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules. Cell. 1989 Nov 3;59(3):421–432. [PubMed]
  • Sluder G, Begg DA. Control mechanisms of the cell cycle: role of the spatial arrangement of spindle components in the timing of mitotic events. J Cell Biol. 1983 Sep;97(3):877–886. [PMC free article] [PubMed]
  • Sluder G, Rieder CL. Centriole number and the reproductive capacity of spindle poles. J Cell Biol. 1985 Mar;100(3):887–896. [PMC free article] [PubMed]
  • Sluder G, Rieder CL. Experimental separation of pronuclei in fertilized sea urchin eggs: chromosomes do not organize a spindle in the absence of centrosomes. J Cell Biol. 1985 Mar;100(3):897–903. [PMC free article] [PubMed]
  • Sluder G, Miller FJ, Rieder CL. The reproduction of centrosomes: nuclear versus cytoplasmic controls. J Cell Biol. 1986 Nov;103(5):1873–1881. [PMC free article] [PubMed]
  • Verde F, Labbé JC, Dorée M, Karsenti E. Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature. 1990 Jan 18;343(6255):233–238. [PubMed]
  • Ward GE, Kirschner MW. Identification of cell cycle-regulated phosphorylation sites on nuclear lamin C. Cell. 1990 May 18;61(4):561–577. [PubMed]
  • Watanabe N, Vande Woude GF, Ikawa Y, Sagata N. Specific proteolysis of the c-mos proto-oncogene product by calpain on fertilization of Xenopus eggs. Nature. 1989 Nov 30;342(6249):505–511. [PubMed]
  • Wordeman L, Cande WZ. Reactivation of spindle elongation in vitro is correlated with the phosphorylation of a 205 kd spindle-associated protein. Cell. 1987 Aug 14;50(4):535–543. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press