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J Cell Biol. 1996 October 2; 135(2): 399–414.
PMCID: PMC2121053

Opposing motor activities are required for the organization of the mammalian mitotic spindle pole

Abstract

We use both in vitro and in vivo approaches to examine the roles of Eg5 (kinesin-related protein), cytoplasmic dynein, and dynactin in the organization of the microtubules and the localization of NuMA (Nu-clear protein that associates with the Mitotic Apparatus) at the polar ends of the mammalian mitotic spindle. Perturbation of the function of Eg5 through either immunodepletion from a cell free system for assembly of mitotic asters or antibody microinjection into cultured cells leads to organized astral microtubule arrays with expanded polar regions in which the minus ends of the microtubules emanate from a ring-like structure that contains NuMA. Conversely, perturbation of the function of cytoplasmic dynein or dynactin through either specific immunodepletition from the cell free system or expression of a dominant negative subunit of dynactin in cultured cells results in the complete lack of organization of microtubules and the failure to efficiently concentrate the NuMA protein despite its association with the microtubules. Simultaneous immunodepletion of these proteins from the cell free system for mitotic aster assembly indicates that the plus end- directed activity of Eg5 antagonizes the minus end-directed activity of cytoplasmic dynein and a minus end-directed activity associated with NuMA during the organization of the microtubules into a morphologic pole. Taken together, these results demonstrate that the unique organization of the minus ends of microtubules and the localization of NuMA at the polar ends of the mammalian mitotic spindle can be accomplished in a centrosome-independent manner by the opposing activities of plus end- and minus end-directed motors.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Afshar K, Barton NR, Hawley RS, Goldstein LS. DNA binding and meiotic chromosomal localization of the Drosophila nod kinesin-like protein. Cell. 1995 Apr 7;81(1):129–138. [PubMed]
  • Ando A, Kikuti YY, Kawata H, Okamoto N, Imai T, Eki T, Yokoyama K, Soeda E, Ikemura T, Abe K, et al. Cloning of a new kinesin-related gene located at the centromeric end of the human MHC region. Immunogenetics. 1994;39(3):194–200. [PubMed]
  • Barton NR, Goldstein LS. Going mobile: microtubule motors and chromosome segregation. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1735–1742. [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]
  • Blangy A, Lane HA, d'Hérin P, Harper M, Kress M, Nigg EA. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell. 1995 Dec 29;83(7):1159–1169. [PubMed]
  • Blose SH, Meltzer DI, Feramisco JR. 10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin. J Cell Biol. 1984 Mar;98(3):847–858. [PMC free article] [PubMed]
  • Burke B, Gerace L. A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell. 1986 Feb 28;44(4):639–652. [PubMed]
  • Capecchi MR. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell. 1980 Nov;22(2 Pt 2):479–488. [PubMed]
  • Clark SW, Meyer DI. ACT3: a putative centractin homologue in S. cerevisiae is required for proper orientation of the mitotic spindle. J Cell Biol. 1994 Oct;127(1):129–138. [PMC free article] [PubMed]
  • Cleveland DW. NuMA: a protein involved in nuclear structure, spindle assembly, and nuclear re-formation. Trends Cell Biol. 1995 Feb;5(2):60–64. [PubMed]
  • Compton DA, Cleveland DW. NuMA is required for the proper completion of mitosis. J Cell Biol. 1993 Feb;120(4):947–957. [PMC free article] [PubMed]
  • Compton DA, Cleveland DW. NuMA, a nuclear protein involved in mitosis and nuclear reformation. Curr Opin Cell Biol. 1994 Jun;6(3):343–346. [PubMed]
  • Compton DA, Luo C. Mutation of the predicted p34cdc2 phosphorylation sites in NuMA impair the assembly of the mitotic spindle and block mitosis. J Cell Sci. 1995 Feb;108(Pt 2):621–633. [PubMed]
  • Compton DA, Yen TJ, Cleveland DW. Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds. J Cell Biol. 1991 Mar;112(6):1083–1097. [PMC free article] [PubMed]
  • Compton DA, Szilak I, Cleveland DW. Primary structure of NuMA, an intranuclear protein that defines a novel pathway for segregation of proteins at mitosis. J Cell Biol. 1992 Mar;116(6):1395–1408. [PMC free article] [PubMed]
  • Coue M, Lombillo VA, McIntosh JR. Microtubule depolymerization promotes particle and chromosome movement in vitro. J Cell Biol. 1991 Mar;112(6):1165–1175. [PMC free article] [PubMed]
  • Desai A, Mitchison TJ. A new role for motor proteins as couplers to depolymerizing microtubules. J Cell Biol. 1995 Jan;128(1-2):1–4. [PMC free article] [PubMed]
  • Doxsey SJ, Stein P, Evans L, Calarco PD, Kirschner M. Pericentrin, a highly conserved centrosome protein involved in microtubule organization. Cell. 1994 Feb 25;76(4):639–650. [PubMed]
  • Earnshaw WC, Tomkiel JE. Centromere and kinetochore structure. Curr Opin Cell Biol. 1992 Feb;4(1):86–93. [PubMed]
  • Echeverri CJ, Paschal BM, Vaughan KT, Vallee RB. Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J Cell Biol. 1996 Feb;132(4):617–633. [PMC free article] [PubMed]
  • Endow SA. Chromosome distribution, molecular motors and the claret protein. Trends Genet. 1993 Feb;9(2):52–55. [PubMed]
  • Endow SA, Chandra R, Komma DJ, Yamamoto AH, Salmon ED. Mutants of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis. J Cell Sci. 1994 Apr;107(Pt 4):859–867. [PubMed]
  • Fuller MT, Wilson PG. Force and counterforce in the mitotic spindle. Cell. 1992 Nov 13;71(4):547–550. [PubMed]
  • Gaglio T, Saredi A, Compton DA. NuMA is required for the organization of microtubules into aster-like mitotic arrays. J Cell Biol. 1995 Nov;131(3):693–708. [PMC free article] [PubMed]
  • Gill SR, Schroer TA, Szilak I, Steuer ER, Sheetz MP, Cleveland DW. Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein. J Cell Biol. 1991 Dec;115(6):1639–1650. [PMC free article] [PubMed]
  • Hayden JH, Bowser SS, Rieder CL. Kinetochores capture astral microtubules during chromosome attachment to the mitotic spindle: direct visualization in live newt lung cells. J Cell Biol. 1990 Sep;111(3):1039–1045. [PMC free article] [PubMed]
  • Holy TE, Leibler S. Dynamic instability of microtubules as an efficient way to search in space. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5682–5685. [PubMed]
  • Hoyt MA. Cellular roles of kinesin and related proteins. Curr Opin Cell Biol. 1994 Feb;6(1):63–68. [PubMed]
  • Hyman AA, Karsenti E. Morphogenetic properties of microtubules and mitotic spindle assembly. Cell. 1996 Feb 9;84(3):401–410. [PubMed]
  • Hyman AA, Mitchison TJ. Two different microtubule-based motor activities with opposite polarities in kinetochores. Nature. 1991 May 16;351(6323):206–211. [PubMed]
  • Inoué S, Salmon ED. Force generation by microtubule assembly/disassembly in mitosis and related movements. Mol Biol Cell. 1995 Dec;6(12):1619–1640. [PMC free article] [PubMed]
  • Kallajoki M, Weber K, Osborn M. A 210 kDa nuclear matrix protein is a functional part of the mitotic spindle; a microinjection study using SPN monoclonal antibodies. EMBO J. 1991 Nov;10(11):3351–3362. [PubMed]
  • Kallajoki M, Weber K, Osborn M. Ability to organize microtubules in taxol-treated mitotic PtK2 cells goes with the SPN antigen and not with the centrosome. J Cell Sci. 1992 May;102(Pt 1):91–102. [PubMed]
  • Kallajoki M, Harborth J, Weber K, Osborn M. Microinjection of a monoclonal antibody against SPN antigen, now identified by peptide sequences as the NuMA protein, induces micronuclei in PtK2 cells. J Cell Sci. 1993 Jan;104(Pt 1):139–150. [PubMed]
  • Karki S, Holzbaur EL. Affinity chromatography demonstrates a direct binding between cytoplasmic dynein and the dynactin complex. J Biol Chem. 1995 Dec 1;270(48):28806–28811. [PubMed]
  • Koshland DE, Mitchison TJ, Kirschner MW. Polewards chromosome movement driven by microtubule depolymerization in vitro. Nature. 1988 Feb 11;331(6156):499–504. [PubMed]
  • Kuriyama R, Kofron M, Essner R, Kato T, Dragas-Granoic S, Omoto CK, Khodjakov A. Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells. J Cell Biol. 1995 May;129(4):1049–1059. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Li X, Nicklas RB. Mitotic forces control a cell-cycle checkpoint. Nature. 1995 Feb 16;373(6515):630–632. [PubMed]
  • Lydersen BK, Pettijohn DE. Human-specific nuclear protein that associates with the polar region of the mitotic apparatus: distribution in a human/hamster hybrid cell. Cell. 1980 Nov;22(2 Pt 2):489–499. [PubMed]
  • Maekawa T, Leslie R, Kuriyama R. Identification of a minus end-specific microtubule-associated protein located at the mitotic poles in cultured mammalian cells. Eur J Cell Biol. 1991 Apr;54(2):255–267. [PubMed]
  • Mastronarde DN, McDonald KL, Ding R, McIntosh JR. Interpolar spindle microtubules in PTK cells. J Cell Biol. 1993 Dec;123(6 Pt 1):1475–1489. [PMC free article] [PubMed]
  • Matthies HJ, McDonald HB, Goldstein LS, Theurkauf WE. Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein. J Cell Biol. 1996 Jul;134(2):455–464. [PMC free article] [PubMed]
  • McIntosh JR, Koonce MP. Mitosis. Science. 1989 Nov 3;246(4930):622–628. [PubMed]
  • McKim KS, Hawley RS. Chromosomal control of meiotic cell division. Science. 1995 Dec 8;270(5242):1595–1601. [PubMed]
  • Mitchison TJ. Mitosis: basic concepts. Curr Opin Cell Biol. 1989 Feb;1(1):67–74. [PubMed]
  • Mitchison TJ. Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence. J Cell Biol. 1989 Aug;109(2):637–652. [PMC free article] [PubMed]
  • Murray AW. Cell cycle. Tense spindles can relax. Nature. 1995 Feb 16;373(6515):560–561. [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, Lee GM, Rieder CL, Rupp G. Mechanically cut mitotic spindles: clean cuts and stable microtubules. J Cell Sci. 1989 Nov;94(Pt 3):415–423. [PubMed]
  • Nicklas RB, Ward SC, Gorbsky GJ. Kinetochore chemistry is sensitive to tension and may link mitotic forces to a cell cycle checkpoint. J Cell Biol. 1995 Aug;130(4):929–939. [PMC free article] [PubMed]
  • Pfarr CM, Coue M, Grissom PM, Hays TS, Porter ME, McIntosh JR. Cytoplasmic dynein is localized to kinetochores during mitosis. Nature. 1990 May 17;345(6272):263–265. [PubMed]
  • Plamann M, Minke PF, Tinsley JH, Bruno KS. Cytoplasmic dynein and actin-related protein Arp1 are required for normal nuclear distribution in filamentous fungi. J Cell Biol. 1994 Oct;127(1):139–149. [PMC free article] [PubMed]
  • Pluta AF, Mackay AM, Ainsztein AM, Goldberg IG, Earnshaw WC. The centromere: hub of chromosomal activities. Science. 1995 Dec 8;270(5242):1591–1594. [PubMed]
  • Rieder CL. The structure of the cold-stable kinetochore fiber in metaphase PtK1 cells. Chromosoma. 1981;84(1):145–158. [PubMed]
  • Rieder CL. Mitosis: towards a molecular understanding of chromosome behavior. Curr Opin Cell Biol. 1991 Feb;3(1):59–66. [PubMed]
  • Rieder CL, Salmon ED. Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle. J Cell Biol. 1994 Feb;124(3):223–233. [PMC free article] [PubMed]
  • Rieder CL, Alexander SP. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells. J Cell Biol. 1990 Jan;110(1):81–95. [PMC free article] [PubMed]
  • Rieder CL, Schultz A, Cole R, Sluder G. Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle. J Cell Biol. 1994 Dec;127(5):1301–1310. [PMC free article] [PubMed]
  • Saredi A, Howard L, Compton DA. NuMA assembles into an extensive filamentous structure when expressed in the cell cytoplasm. J Cell Sci. 1996 Mar;109(Pt 3):619–630. [PubMed]
  • Saunders WS, Hoyt MA. Kinesin-related proteins required for structural integrity of the mitotic spindle. Cell. 1992 Aug 7;70(3):451–458. [PubMed]
  • Saunders WS, Koshland D, Eshel D, Gibbons IR, Hoyt MA. Saccharomyces cerevisiae kinesin- and dynein-related proteins required for anaphase chromosome segregation. J Cell Biol. 1995 Feb;128(4):617–624. [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]
  • Sawin KE, LeGuellec K, Philippe M, Mitchison TJ. Mitotic spindle organization by a plus-end-directed microtubule motor. Nature. 1992 Oct 8;359(6395):540–543. [PubMed]
  • Sawin KE, Mitchison TJ, Wordeman LG. Evidence for kinesin-related proteins in the mitotic apparatus using peptide antibodies. J Cell Sci. 1992 Feb;101(Pt 2):303–313. [PubMed]
  • Schafer DA, Gill SR, Cooper JA, Heuser JE, Schroer TA. Ultrastructural analysis of the dynactin complex: an actin-related protein is a component of a filament that resembles F-actin. J Cell Biol. 1994 Jul;126(2):403–412. [PMC free article] [PubMed]
  • Schroer TA. Structure, function and regulation of cytoplasmic dynein. Curr Opin Cell Biol. 1994 Feb;6(1):69–73. [PubMed]
  • Schroer TA. New insights into the interaction of cytoplasmic dynein with the actin-related protein, Arp1. J Cell Biol. 1994 Oct;127(1):1–4. [PMC free article] [PubMed]
  • Schroer TA, Sheetz MP. Two activators of microtubule-based vesicle transport. J Cell Biol. 1991 Dec;115(5):1309–1318. [PMC free article] [PubMed]
  • Schroer TA, Bingham JB, Gill SR. Actin-related protein 1 and cytoplasmic dynein-based motility - what's the connection? Trends Cell Biol. 1996 Jun;6(6):212–215. [PubMed]
  • Steuer ER, Wordeman L, Schroer TA, Sheetz MP. Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature. 1990 May 17;345(6272):266–268. [PubMed]
  • Theurkauf WE, Hawley RS. Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein. J Cell Biol. 1992 Mar;116(5):1167–1180. [PMC free article] [PubMed]
  • Tinsley JH, Minke PF, Bruno KS, Plamann M. p150Glued, the largest subunit of the dynactin complex, is nonessential in Neurospora but required for nuclear distribution. Mol Biol Cell. 1996 May;7(5):731–742. [PMC free article] [PubMed]
  • Tousson A, Zeng C, Brinkley BR, Valdivia MM. Centrophilin: a novel mitotic spindle protein involved in microtubule nucleation. J Cell Biol. 1991 Feb;112(3):427–440. [PMC free article] [PubMed]
  • Vaisberg EA, Grissom PM, McIntosh JR. Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles. J Cell Biol. 1996 May;133(4):831–842. [PMC free article] [PubMed]
  • Vallee RB, Sheetz MP. Targeting of motor proteins. Science. 1996 Mar 15;271(5255):1539–1544. [PubMed]
  • Vaughan KT, Vallee RB. Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued. J Cell Biol. 1995 Dec;131(6 Pt 1):1507–1516. [PMC free article] [PubMed]
  • Verde F, Berrez JM, Antony C, Karsenti E. Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs: requirement for phosphorylated factors and cytoplasmic dynein. J Cell Biol. 1991 Mar;112(6):1177–1187. [PMC free article] [PubMed]
  • Vernos I, Karsenti E. Chromosomes take the lead in spindle assembly. Trends Cell Biol. 1995 Aug;5(8):297–301. [PubMed]
  • Vernos I, Raats J, Hirano T, Heasman J, Karsenti E, Wylie C. Xklp1, a chromosomal Xenopus kinesin-like protein essential for spindle organization and chromosome positioning. Cell. 1995 Apr 7;81(1):117–127. [PubMed]
  • Walczak CE, Mitchison TJ. Kinesin-related proteins at mitotic spindle poles: function and regulation. Cell. 1996 Jun 28;85(7):943–946. [PubMed]
  • Wang SZ, Adler R. Chromokinesin: a DNA-binding, kinesin-like nuclear protein. J Cell Biol. 1995 Mar;128(5):761–768. [PMC free article] [PubMed]
  • Waterman-Storer CM, Karki S, Holzbaur EL. The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1). Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1634–1638. [PubMed]
  • Waters JC, Cole RW, Rieder CL. The force-producing mechanism for centrosome separation during spindle formation in vertebrates is intrinsic to each aster. J Cell Biol. 1993 Jul;122(2):361–372. [PMC free article] [PubMed]
  • Yang CH, Snyder M. The nuclear-mitotic apparatus protein is important in the establishment and maintenance of the bipolar mitotic spindle apparatus. Mol Biol Cell. 1992 Nov;3(11):1259–1267. [PMC free article] [PubMed]
  • Yang CH, Lambie EJ, Snyder M. NuMA: an unusually long coiled-coil related protein in the mammalian nucleus. J Cell Biol. 1992 Mar;116(6):1303–1317. [PMC free article] [PubMed]

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