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J Cell Biol. Dec 1, 1989; 109(6): 2977–2991.
PMCID: PMC2115930

Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo

Abstract

We have developed affinity chromatography methods for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts and have used them to analyze the cytoskeleton of the early Drosophila embryo. More than 50 Drosophila embryo proteins bind to microtubule affinity columns. To begin to characterize these proteins, we have generated individual mouse polyclonal antibodies that specifically recognize 24 of them. As judged by immunofluorescence, some of the antigens localize to the mitotic spindle in the early Drosophila embryo, while others are present in centrosomes, kinetochores, subsets of microtubules, or a combination of these structures. Since 20 of the 24 antibodies stain microtubule structures, it is likely that most of the proteins that bind to our columns are associated with microtubules in vivo. Very few MAPS seem to be identically localized in the cell, indicating that the microtubule cytoskeleton is remarkably complex.

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Selected References

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  • Binder LI, Frankfurter A, Rebhun LI. The distribution of tau in the mammalian central nervous system. J Cell Biol. 1985 Oct;101(4):1371–1378. [PMC free article] [PubMed]
  • Bloom GS, Luca FC, Vallee RB. Widespread cellular distribution of MAP-1A (microtubule-associated protein 1A) in the mitotic spindle and on interphase microtubules. J Cell Biol. 1984 Jan;98(1):331–340. [PMC free article] [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]
  • Burke B, Griffiths G, Reggio H, Louvard D, Warren G. A monoclonal antibody against a 135-K Golgi membrane protein. EMBO J. 1982;1(12):1621–1628. [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]
  • Dedhar S, Ruoslahti E, Pierschbacher MD. A cell surface receptor complex for collagen type I recognizes the Arg-Gly-Asp sequence. J Cell Biol. 1987 Mar;104(3):585–593. [PMC free article] [PubMed]
  • Detrich HW, 3rd, Wilson L. Purification, characterization, and assembly properties of tubulin from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. Biochemistry. 1983 May 10;22(10):2453–2462. [PubMed]
  • Foe VE, Alberts BM. Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. J Cell Sci. 1983 May;61:31–70. [PubMed]
  • Gard DL, Kirschner MW. A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line. J Cell Biol. 1985 Mar;100(3):764–774. [PMC free article] [PubMed]
  • Goldstein LS, Laymon RA, McIntosh JR. A microtubule-associated protein in Drosophila melanogaster: identification, characterization, and isolation of coding sequences. J Cell Biol. 1986 Jun;102(6):2076–2087. [PMC free article] [PubMed]
  • Gosti-Testu F, Marty MC, Berges J, Maunoury R, Bornens M. Identification of centrosomal proteins in a human lymphoblastic cell line. EMBO J. 1986 Oct;5(10):2545–2550. [PMC free article] [PubMed]
  • Gotlieb AI, May LM, Subrahmanyan L, Kalnins VI. Distribution of microtubule organizing centers in migrating sheets of endothelial cells. J Cell Biol. 1981 Nov;91(2 Pt 1):589–594. [PMC free article] [PubMed]
  • Horwitz SB, Parness J, Schiff PB, Manfredi JJ. Taxol: a new probe for studying the structure and function of microtubules. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 1):219–226. [PubMed]
  • Huber G, Matus A. Differences in the cellular distributions of two microtubule-associated proteins, MAP1 and MAP2, in rat brain. J Neurosci. 1984 Jan;4(1):151–160. [PubMed]
  • Karr TL, Alberts BM. Organization of the cytoskeleton in early Drosophila embryos. J Cell Biol. 1986 Apr;102(4):1494–1509. [PMC free article] [PubMed]
  • Kellogg DR, Mitchison TJ, Alberts BM. Behaviour of microtubules and actin filaments in living Drosophila embryos. Development. 1988 Aug;103(4):675–686. [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]
  • 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]
  • Mabuchi I, Okuno M. The effect of myosin antibody on the division of starfish blastomeres. J Cell Biol. 1977 Jul;74(1):251–263. [PMC free article] [PubMed]
  • Miller KG, Alberts BM. F-actin affinity chromatography: technique for isolating previously unidentified actin-binding proteins. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4808–4812. [PMC free article] [PubMed]
  • Miller KG, Field CM, Alberts BM. Actin-binding proteins from Drosophila embryos: a complex network of interacting proteins detected by F-actin affinity chromatography. J Cell Biol. 1989 Dec;109(6 Pt 1):2963–2975. [PMC free article] [PubMed]
  • Mitchison T, Kirschner M. Microtubule assembly nucleated by isolated centrosomes. Nature. 1984 Nov 15;312(5991):232–237. [PubMed]
  • Moroi Y, Murata I, Takeuchi A, Kamatani N, Tanimoto K, Yokohari R. Human anticentriole autoantibody in patients with scleroderma and Raynaud's phenomenon. Clin Immunol Immunopathol. 1983 Dec;29(3):381–390. [PubMed]
  • Raff JW, Glover DM. Nuclear and cytoplasmic mitotic cycles continue in Drosophila embryos in which DNA synthesis is inhibited with aphidicolin. J Cell Biol. 1988 Dec;107(6 Pt 1):2009–2019. [PMC free article] [PubMed]
  • Schiff PB, Fant J, Horwitz SB. Promotion of microtubule assembly in vitro by taxol. Nature. 1979 Feb 22;277(5698):665–667. [PubMed]
  • Smith DE, Fisher PA. Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol. 1984 Jul;99(1 Pt 1):20–28. [PMC free article] [PubMed]
  • Theurkauf WE, Baum H, Bo J, Wensink PC. Tissue-specific and constitutive alpha-tubulin genes of Drosophila melanogaster code for structurally distinct proteins. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8477–8481. [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]
  • Vallee RB, Bloom GS. Isolation of sea urchin egg microtubules with taxol and identification of mitotic spindle microtubule-associated proteins with monoclonal antibodies. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6259–6263. [PMC free article] [PubMed]
  • Vallee RB, Collins CA. Purification of microtubules and microtubule-associated proteins from sea urchin eggs and cultured mammalian cells using taxol, and use of exogenous taxol-stabilized brain microtubules for purifying microtubule-associated proteins. Methods Enzymol. 1986;134:116–127. [PubMed]
  • Vallee RB, Bloom GS, Theurkauf WE. Microtubule-associated proteins: subunits of the cytomatrix. J Cell Biol. 1984 Jul;99(1 Pt 2):38s–44s. [PMC free article] [PubMed]
  • Vorobjev IA, Nadezhdina ES. The centrosome and its role in the organization of microtubules. Int Rev Cytol. 1987;106:227–293. [PubMed]
  • Vorobjev IA, Chentsov YuS Centrioles in the cell cycle. I. Epithelial cells. J Cell Biol. 1982 Jun;93(3):938–949. [PMC free article] [PubMed]
  • Warn RM, Warn A. Microtubule arrays present during the syncytial and cellular blastoderm stages of the early Drosophila embryo. Exp Cell Res. 1986 Mar;163(1):201–210. [PubMed]
  • Warn RM, Flegg L, Warn A. An investigation of microtubule organization and functions in living Drosophila embryos by injection of a fluorescently labeled antibody against tyrosinated alpha-tubulin. J Cell Biol. 1987 Oct;105(4):1721–1730. [PMC free article] [PubMed]
  • Weingarten MD, Suter MM, Littman DR, Kirschner MW. Properties of the depolymerization products of microtubules from mammalian brain. Biochemistry. 1974 Dec 31;13(27):5529–5537. [PubMed]
  • Whitfield WG, Millar SE, Saumweber H, Frasch M, Glover DM. Cloning of a gene encoding an antigen associated with the centrosome in Drosophila. J Cell Sci. 1988 Apr;89(Pt 4):467–480. [PubMed]
  • Wilson L. Properties of colchicine binding protein from chick embryo brain. Interactions with vinca alkaloids and podophyllotoxin. Biochemistry. 1970 Dec 8;9(25):4999–5007. [PubMed]

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