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J Cell Biol. Dec 1, 1991; 115(5): 1309–1318.
PMCID: PMC2289226

Two activators of microtubule-based vesicle transport

Abstract

Cytoplasmic dynein purified by nucleotide dependent microtubule affinity has significant minus end-directed vesicle motor activity that decreases with each further purification step. Highly purified dynein causes membrane vesicles to bind but not move on microtubules. We exploited these observations to develop an assay for factors that, in combination with dynein, would permit minus end-directed vesicle motility. At each step of the purification, non-dynein fractions were recombined with dynein and assayed for vesicle motility. Two activating fractions were identified by this method. One, called Activator I, copurified with 20S dynein by velocity sedimentation but could be separated from it by ion exchange chromatography. Activator I increased only the frequency of dynein-driven vesicle movements. Activator II, sedimenting at 9S, increased both the frequency and velocity of vesicle transport and also supported plus end movements. Our results suggest that dynein-based motility is controlled at multiple levels and provide a preliminary characterization of two regulatory factors.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Allan VJ, Vale RD. Cell cycle control of microtubule-based membrane transport and tubule formation in vitro. J Cell Biol. 1991 Apr;113(2):347–359. [PMC free article] [PubMed]
  • Amos LA. Brain dynein crossbridges microtubules into bundles. J Cell Sci. 1989 May;93(Pt 1):19–28. [PubMed]
  • Bomsel M, Parton R, Kuznetsov SA, Schroer TA, Gruenberg J. Microtubule- and motor-dependent fusion in vitro between apical and basolateral endocytic vesicles from MDCK cells. Cell. 1990 Aug 24;62(4):719–731. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Brady ST, Lasek RJ, Allen RD. Video microscopy of fast axonal transport in extruded axoplasm: a new model for study of molecular mechanisms. Cell Motil. 1985;5(2):81–101. [PubMed]
  • Cohn SA, Ingold AL, Scholey JM. Correlation between the ATPase and microtubule translocating activities of sea urchin egg kinesin. Nature. 1987 Jul 9;328(6126):160–163. [PubMed]
  • Collins CA, Vallee RB. Preparation of microtubules from rat liver and testis: cytoplasmic dynein is a major microtubule associated protein. Cell Motil Cytoskeleton. 1989;14(4):491–500. [PubMed]
  • Dabora SL, Sheetz MP. Cultured cell extracts support organelle movement on microtubules in vitro. Cell Motil Cytoskeleton. 1988;10(4):482–495. [PubMed]
  • Euteneuer U, Koonce MP, Pfister KK, Schliwa M. An ATPase with properties expected for the organelle motor of the giant amoeba, Reticulomyxa. Nature. 1988 Mar 10;332(6160):176–178. [PubMed]
  • Gibbons IR, Lee-Eiford A, Mocz G, Phillipson CA, Tang WJ, Gibbons BH. Photosensitized cleavage of dynein heavy chains. Cleavage at the "V1 site" by irradiation at 365 nm in the presence of ATP and vanadate. J Biol Chem. 1987 Feb 25;262(6):2780–2786. [PubMed]
  • Gilbert SP, Sloboda RD. A squid dynein isoform promotes axoplasmic vesicle translocation. J Cell Biol. 1989 Nov;109(5):2379–2394. [PMC free article] [PubMed]
  • Gruenberg J, Griffiths G, Howell KE. Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. J Cell Biol. 1989 Apr;108(4):1301–1316. [PMC free article] [PubMed]
  • Hirokawa N, Pfister KK, Yorifuji H, Wagner MC, Brady ST, Bloom GS. Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration. Cell. 1989 Mar 10;56(5):867–878. [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]
  • Holzbaur EL, Hammarback JA, Paschal BM, Kravit NG, Pfister KK, Vallee RB. Homology of a 150K cytoplasmic dynein-associated polypeptide with the Drosophila gene Glued. Nature. 1991 Jun 13;351(6327):579–583. [PubMed]
  • Ingold AL, Cohn SA, Scholey JM. Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains. J Cell Biol. 1988 Dec;107(6 Pt 2):2657–2667. [PMC free article] [PubMed]
  • Koonce MP, McIntosh JR. Identification and immunolocalization of cytoplasmic dynein in Dictyostelium. Cell Motil Cytoskeleton. 1990;15(1):51–62. [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]
  • Lessard JL. Two monoclonal antibodies to actin: one muscle selective and one generally reactive. Cell Motil Cytoskeleton. 1988;10(3):349–362. [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]
  • Moreno S, Nurse P. Substrates for p34cdc2: in vivo veritas? Cell. 1990 May 18;61(4):549–551. [PubMed]
  • Neely MD, Boekelheide K. Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule-associated protein (cytoplasmic dynein). J Cell Biol. 1988 Nov;107(5):1767–1776. [PMC free article] [PubMed]
  • Paschal BM, Vallee RB. Retrograde transport by the microtubule-associated protein MAP 1C. Nature. 1987 Nov 12;330(6144):181–183. [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]
  • Rozdzial MM, Haimo LT. Bidirectional pigment granule movements of melanophores are regulated by protein phosphorylation and dephosphorylation. Cell. 1986 Dec 26;47(6):1061–1070. [PubMed]
  • Sale WS, Satir P. Direction of active sliding of microtubules in Tetrahymena cilia. Proc Natl Acad Sci U S A. 1977 May;74(5):2045–2049. [PMC free article] [PubMed]
  • Schaffner W, Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. [PubMed]
  • Schnapp BJ. Viewing single microtubules by video light microscopy. Methods Enzymol. 1986;134:561–573. [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. [PMC free article] [PubMed]
  • Schroer TA, Sheetz MP. Functions of microtubule-based motors. Annu Rev Physiol. 1991;53:629–652. [PubMed]
  • Schroer TA, Schnapp BJ, Reese TS, Sheetz MP. The role of kinesin and other soluble factors in organelle movement along microtubules. J Cell Biol. 1988 Nov;107(5):1785–1792. [PMC free article] [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]
  • Sheetz MP, Block SM, Spudich JA. Myosin movement in vitro: a quantitative assay using oriented actin cables from Nitella. Methods Enzymol. 1986;134:531–544. [PubMed]
  • Sheetz MP, Steuer ER, Schroer TA. The mechanism and regulation of fast axonal transport. Trends Neurosci. 1989 Nov;12(11):474–478. [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]
  • Tash JS. Protein phosphorylation: the second messenger signal transducer of flagellar motility. Cell Motil Cytoskeleton. 1989;14(3):332–339. [PubMed]
  • Thaler CD, Haimo LT. Regulation of organelle transport in melanophores by calcineurin. J Cell Biol. 1990 Nov;111(5 Pt 1):1939–1948. [PMC free article] [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PMC free article] [PubMed]
  • Vale RD, Szent-Gyorgyi AG, Sheetz MP. Movement of scallop myosin on Nitella actin filaments: regulation by calcium. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6775–6778. [PMC free article] [PubMed]
  • Vale RD, Reese TS, Sheetz MP. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell. 1985 Aug;42(1):39–50. [PMC free article] [PubMed]
  • Vale RD, Soll DR, Gibbons IR. One-dimensional diffusion of microtubules bound to flagellar dynein. Cell. 1989 Dec 1;59(5):915–925. [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]
  • Williams RC, Jr, Lee JC. Preparation of tubulin from brain. Methods Enzymol. 1982;85(Pt B):376–385. [PubMed]
  • Wordeman L, Steuer ER, Sheetz MP, Mitchison T. Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes. J Cell Biol. 1991 Jul;114(2):285–294. [PMC free article] [PubMed]
  • Yang JT, Laymon RA, Goldstein LS. A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses. Cell. 1989 Mar 10;56(5):879–889. [PubMed]

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