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. 1978 Mar 1;76(3):781–786. doi: 10.1083/jcb.76.3.781

Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence

PMCID: PMC2110001  PMID: 344329

Abstract

Microtubule accessory proteins were isolated from porcine brain microtubules by phosphocellulose chromatography, and the high molecular weight protein (HMW protein), purified from this microtubule-associated fraction by electrophoretic elution from SDS gels, was used to raise antisera in rabbits. In agarose double diffusion tests, the antiserum obtained forms precipitin lines with purified HMW protein but not with tau protein or tubulin. When rat glial cells (strain C6) are examined by indirect immunofluorescence, this serum specifically stains a colchicine-sensitive filamentous cytoplasmic network in interphase cells, a network indistinguishable from that seen when cells are treated with antitubulin serum. In dividing cells, specific staining of the mitotic spindle and the stem body is observed with the antiserum to HMW protein. These studies indicate that HMW protein, like tau protein, is associated with microtubules in intact cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aubin J. E., Subrahmanyan L., Kalnins V. I., Ling V. Antisera against electrophoretically purified tubulin stimulate colchicine-binding activity. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1246–1249. doi: 10.1073/pnas.73.4.1246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cleveland D. W., Hwo S. Y., Kirschner M. W. Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly. J Mol Biol. 1977 Oct 25;116(2):227–247. doi: 10.1016/0022-2836(77)90214-5. [DOI] [PubMed] [Google Scholar]
  3. Cleveland D. W., Hwo S. Y., Kirschner M. W. Purification of tau, a microtubule-associated protein that induces assembly of microtubules from purified tubulin. J Mol Biol. 1977 Oct 25;116(2):207–225. doi: 10.1016/0022-2836(77)90213-3. [DOI] [PubMed] [Google Scholar]
  4. Connolly J. A., Kalnins V. I., Cleveland D. W., Kirschner M. W. Immunoflourescent staining of cytoplasmic and spindle microtubules in mouse fibroblasts with antibody to tau protein. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2437–2440. doi: 10.1073/pnas.74.6.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Erickson H. P., Voter W. A. Polycation-induced assembly of purified tubulin. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2813–2817. doi: 10.1073/pnas.73.8.2813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Forer A., Kalnins V. I., Zimmerman A. M. Spindle birefringence of isolated mitotic apparatus: further evidence for two birefringent spindle components. J Cell Sci. 1976 Oct;22(1):115–131. doi: 10.1242/jcs.22.1.115. [DOI] [PubMed] [Google Scholar]
  7. Herzog W., Weber K. In vitro assembly of pure tubulin into microtubules in the absence of microtubule-associated proteins and glycerol. Proc Natl Acad Sci U S A. 1977 May;74(5):1860–1864. doi: 10.1073/pnas.74.5.1860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Himes R. H., Burton P. R., Kersey R. N., Pierson G. B. Brain tubulin polymerization in the absence of "microtubule-associated proteins". Proc Natl Acad Sci U S A. 1976 Dec;73(12):4397–4399. doi: 10.1073/pnas.73.12.4397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jorgensen A. O., Subrahmanyan L., Turnbull C., Kalnins V. I. Localization of the neurofilament protein in neuroblastoma cells by immunofluorescent staining. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3192–3196. doi: 10.1073/pnas.73.9.3192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Keates R. A., Hall R. H. Tubulin requires an accessory protein for self assembly in microtubules. Nature. 1975 Oct 2;257(5525):418–421. doi: 10.1038/257418a0. [DOI] [PubMed] [Google Scholar]
  11. Kirschner M. W., Williams R. C., Weingarten M., Gerhart J. C. Microtubules from mammalian brain: some properties of their depolymerization products and a proposed mechanism of assembly and disassembly. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1159–1163. doi: 10.1073/pnas.71.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lee J. C., Timasheff S. N. The reconstitution of microtubules from purified calf brain tubulin. Biochemistry. 1975 Nov 18;14(23):5183–5187. doi: 10.1021/bi00694a025. [DOI] [PubMed] [Google Scholar]
  13. Murphy D. B., Borisy G. G. Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2696–2700. doi: 10.1073/pnas.72.7.2696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Murphy D. B., Vallee R. B., Borisy G. G. Identity and polymerization-stimulatory activity of the nontubulin proteins associated with microtubules. Biochemistry. 1977 Jun 14;16(12):2598–2605. doi: 10.1021/bi00631a004. [DOI] [PubMed] [Google Scholar]
  15. Osborn M., Franke W. W., Weber K. Visualization of a system of filaments 7-10 nm thick in cultured cells of an epithelioid line (Pt K2) by immunofluorescence microscopy. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2490–2494. doi: 10.1073/pnas.74.6.2490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sloboda R. D., Dentler W. L., Rosenbaum J. L. Microtubule-associated proteins and the stimulation of tubulin assembly in vitro. Biochemistry. 1976 Oct 5;15(20):4497–4505. doi: 10.1021/bi00665a026. [DOI] [PubMed] [Google Scholar]
  18. Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Weingarten M. D., Suter M. M., Littman D. R., Kirschner M. W. Properties of the depolymerization products of microtubules from mammalian brain. Biochemistry. 1974 Dec 31;13(27):5529–5537. doi: 10.1021/bi00724a012. [DOI] [PubMed] [Google Scholar]
  20. Wiche G., Cole R. D. Reversible in vitro polymerization of tubulin from a cultured cell line (rat glial cell clone C6). Proc Natl Acad Sci U S A. 1976 Apr;73(4):1227–1231. doi: 10.1073/pnas.73.4.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Witman G. B., Cleveland D. W., Weingarten M. D., Kirschner M. W. Tubulin requires tau for growth onto microtubule initiating sites. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4070–4074. doi: 10.1073/pnas.73.11.4070. [DOI] [PMC free article] [PubMed] [Google Scholar]

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