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J Cell Biol. 1993 November 2; 123(4): 849–858.
PMCID: PMC2200153

Cytoplasmic dynein plays a role in mammalian mitotic spindle formation

Abstract

The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles, but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP- hydrolytic sites of the cytoplasmic dynein heavy chain from HeLa cells and from Dictyostelium. Monospecific antibodies have been raised to the resulting polypeptides, and these inhibit dynein motor activity in vitro. Their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells become arrested with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles that it may play are redundant. Possible mechanisms of dynein's involvement in mitosis are discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Aist JR, Bayles CJ, Tao W, Berns MW. Direct experimental evidence for the existence, structural basis and function of astral forces during anaphase B in vivo. J Cell Sci. 1991 Oct;100(Pt 2):279–288. [PubMed]
  • 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]
  • Calarco-Gillam PD, Siebert MC, Hubble R, Mitchison T, Kirschner M. Centrosome development in early mouse embryos as defined by an autoantibody against pericentriolar material. Cell. 1983 Dec;35(3 Pt 2):621–629. [PubMed]
  • Endow SA, Titus MA. Genetic approaches to molecular motors. Annu Rev Cell Biol. 1992;8:29–66. [PubMed]
  • Fuller MT, Wilson PG. Force and counterforce in the mitotic spindle. Cell. 1992 Nov 13;71(4):547–550. [PubMed]
  • Gelfand VI, Scholey JM. Cell biology. Every motion has its motor. Nature. 1992 Oct 8;359(6395):480–482. [PubMed]
  • Gibbons IR, Gibbons BH, Mocz G, Asai DJ. Multiple nucleotide-binding sites in the sequence of dynein beta heavy chain. Nature. 1991 Aug 15;352(6336):640–643. [PubMed]
  • Hiramoto Y, Nakano Y. Micromanipulation studies of the mitotic apparatus in sand dollar eggs. Cell Motil Cytoskeleton. 1988;10(1-2):172–184. [PubMed]
  • Hirokawa N, Sato-Yoshitake R, Yoshida T, Kawashima T. Brain dynein (MAP1C) localizes on both anterogradely and retrogradely transported membranous organelles in vivo. J Cell Biol. 1990 Sep;111(3):1027–1037. [PMC free article] [PubMed]
  • Hollenbeck PJ. The distribution, abundance and subcellular localization of kinesin. J Cell Biol. 1989 Jun;108(6):2335–2342. [PMC free article] [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]
  • Hyman AA, Middleton K, Centola M, Mitchison TJ, Carbon J. Microtubule-motor activity of a yeast centromere-binding protein complex. Nature. 1992 Oct 8;359(6395):533–536. [PubMed]
  • Koonce MP, Grissom PM, McIntosh JR. Dynein from Dictyostelium: primary structure comparisons between a cytoplasmic motor enzyme and flagellar dynein. J Cell Biol. 1992 Dec;119(6):1597–1604. [PMC free article] [PubMed]
  • Lacey ML, Haimo LT. Cytoplasmic dynein is a vesicle protein. J Biol Chem. 1992 Mar 5;267(7):4793–4798. [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]
  • Lin KH, Cheng SY. An efficient method to purify active eukaryotic proteins from the inclusion bodies in Escherichia coli. Biotechniques. 1991 Dec;11(6):748–753. [PubMed]
  • Lin SX, Collins CA. Immunolocalization of cytoplasmic dynein to lysosomes in cultured cells. J Cell Sci. 1992 Jan;101(Pt 1):125–137. [PubMed]
  • Lye RJ, Porter ME, Scholey JM, McIntosh JR. Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans. Cell. 1987 Oct 23;51(2):309–318. [PubMed]
  • McDonald HB, Stewart RJ, Goldstein LS. The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor. Cell. 1990 Dec 21;63(6):1159–1165. [PubMed]
  • McIntosh JR, Hering GE. Spindle fiber action and chromosome movement. Annu Rev Cell Biol. 1991;7:403–426. [PubMed]
  • McIntosh JR, Pfarr CM. Mitotic motors. J Cell Biol. 1991 Nov;115(3):577–585. [PMC free article] [PubMed]
  • McIntosh JR, Porter ME. Enzymes for microtubule-dependent motility. J Biol Chem. 1989 Apr 15;264(11):6001–6004. [PubMed]
  • Mikami A, Paschal BM, Mazumdar M, Vallee RB. Molecular cloning of the retrograde transport motor cytoplasmic dynein (MAP 1C). Neuron. 1993 May;10(5):787–796. [PubMed]
  • Nislow C, Sellitto C, Kuriyama R, McIntosh JR. A monoclonal antibody to a mitotic microtubule-associated protein blocks mitotic progression. J Cell Biol. 1990 Aug;111(2):511–522. [PMC free article] [PubMed]
  • Ogawa K. Four ATP-binding sites in the midregion of the beta heavy chain of dynein. Nature. 1991 Aug 15;352(6336):643–645. [PubMed]
  • Paschal BM, Shpetner HS, Vallee RB. MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties. J Cell Biol. 1987 Sep;105(3):1273–1282. [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]
  • 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]
  • Saunders WS, Hoyt MA. Kinesin-related proteins required for structural integrity of the mitotic spindle. Cell. 1992 Aug 7;70(3):451–458. [PubMed]
  • Sawin KE, Scholey JM. Motor proteins in cell division. Trends Cell Biol. 1991 Nov;1(5):122–129. [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]
  • Schnapp BJ, Reese TS. Dynein is the motor for retrograde axonal transport of organelles. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1548–1552. [PubMed]
  • Schroer TA, Steuer ER, Sheetz MP. Cytoplasmic dynein is a minus end-directed motor for membranous organelles. Cell. 1989 Mar 24;56(6):937–946. [PubMed]
  • Sorokin SP. Reconstructions of centriole formation and ciliogenesis in mammalian lungs. J Cell Sci. 1968 Jun;3(2):207–230. [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]
  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. [PubMed]
  • Vale RD, Schnapp BJ, Reese TS, Sheetz MP. Movement of organelles along filaments dissociated from the axoplasm of the squid giant axon. Cell. 1985 Feb;40(2):449–454. [PubMed]
  • Vallee RB, Shpetner HS. Motor proteins of cytoplasmic microtubules. Annu Rev Biochem. 1990;59:909–932. [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]
  • Walker RA, Salmon ED, Endow SA. The Drosophila claret segregation protein is a minus-end directed motor molecule. Nature. 1990 Oct 25;347(6295):780–782. [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]
  • Zavortink M, Welsh MJ, McIntosh JR. The distribution of calmodulin in living mitotic cells. Exp Cell Res. 1983 Dec;149(2):375–385. [PubMed]

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