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. 1985 Nov;4(11):2807–2814. doi: 10.1002/j.1460-2075.1985.tb04007.x

Disruption of microtubules in living cells and cell models by high affinity antibodies to beta-tubulin.

A Füchtbauer, M Herrmann, E M Mandelkow, B M Jockusch
PMCID: PMC554582  PMID: 3905386

Abstract

Polyclonal antibodies with high affinity for beta-tubulin were found to disrupt cytoplasmic microtubules efficiently after microinjection into tissue culture cells. The degree of microtubular fragmentation was directly proportional to the amount of the injected antibody. At molar ratios of 1 antibody per 100 tubulin dimers, most microtubules were disrupted within 90 min after injection. In contrast, the time course of disintegration was relatively independent of the antibody concentration. Within the range of 1 antibody per 10(2)-10(4) tubulin dimers, the maximal values for microtubular disintegration were reached approximately 1-1.5 h after injection. Mitotic microtubules were found to be resistant to all antibody concentrations used. In living cells, microtubules recovered within a few hours after antibody-induced decay. The time course of recovery, like the extent of disintegration, was a function of the antibody concentration. The antibody acted also on microtubules in detergent-extracted cell models and on microtubules polymerised in vitro. When added to microtubular protein, the bivalent antibody as well as its Fab fragments prevented polymerisation. The data suggest that these antibodies disrupt microtubules because their affinity to tubulin is at least 100 times higher than the affinities found for tubulin:tubulin interaction. Fragmented microtubules are probably unstable and decompose into smaller units.

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Selected References

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

  1. Blose S. H., Meltzer D. I., Feramisco J. R. 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. doi: 10.1083/jcb.98.3.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Borisy G. G., Marcum J. M., Olmsted J. B., Murphy D. B., Johnson K. A. Purification of tubulin and associated high molecular weight proteins from porcine brain and characterization of microtubule assembly in vitro. Ann N Y Acad Sci. 1975 Jun 30;253:107–132. doi: 10.1111/j.1749-6632.1975.tb19196.x. [DOI] [PubMed] [Google Scholar]
  3. Brandtzaeg P. Conjugates of immunoglobulin G with different fluorochromes. I. Characterization by anionic-exchange chromatography. Scand J Immunol. 1973;2(3):273–290. doi: 10.1111/j.1365-3083.1973.tb02037.x. [DOI] [PubMed] [Google Scholar]
  4. Bretscher A., Weber K. Villin: the major microfilament-associated protein of the intestinal microvillus. Proc Natl Acad Sci U S A. 1979 May;76(5):2321–2325. doi: 10.1073/pnas.76.5.2321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chaponnier C., Patebex P., Gabbiani G. Human plasma actin-depolymerizing factor. Purification, biological activity and localization in leukocytes and platelets. Eur J Biochem. 1985 Jan 15;146(2):267–276. doi: 10.1111/j.1432-1033.1985.tb08649.x. [DOI] [PubMed] [Google Scholar]
  6. Füchtbauer A., Jockusch B. M., Maruta H., Kilimann M. W., Isenberg G. Disruption of microfilament organization after injection of F-actin capping proteins into living tissue culture cells. 1983 Jul 28-Aug 3Nature. 304(5924):361–364. doi: 10.1038/304361a0. [DOI] [PubMed] [Google Scholar]
  7. Graessmann M., Graessman A. "Early" simian-virus-40-specific RNA contains information for tumor antigen formation and chromatin replication. Proc Natl Acad Sci U S A. 1976 Feb;73(2):366–370. doi: 10.1073/pnas.73.2.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jockusch B. M. Patterns of microfilament organization in animal cells. Mol Cell Endocrinol. 1983 Jan;29(1):1–19. doi: 10.1016/0303-7207(83)90002-3. [DOI] [PubMed] [Google Scholar]
  9. Kilmartin J. V., Wright B., Milstein C. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J Cell Biol. 1982 Jun;93(3):576–582. doi: 10.1083/jcb.93.3.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kirchner K., Mandelkow E. M. Tubulin domains responsible for assembly of dimers and protofilaments. EMBO J. 1985 Sep;4(9):2397–2402. doi: 10.1002/j.1460-2075.1985.tb03945.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kreis T. E., Birchmeier W. Microinjection of fluorescently labeled proteins into living cells with emphasis on cytoskeletal proteins. Int Rev Cytol. 1982;75:209–214. doi: 10.1016/s0074-7696(08)61005-0. [DOI] [PubMed] [Google Scholar]
  12. Mandelkow E. M., Herrmann M., Rühl U. Tubulin domains probed by limited proteolysis and subunit-specific antibodies. J Mol Biol. 1985 Sep 20;185(2):311–327. doi: 10.1016/0022-2836(85)90406-1. [DOI] [PubMed] [Google Scholar]
  13. Osborn M., Webster R. E., Weber K. Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell. J Cell Biol. 1978 Jun;77(3):R27–R34. doi: 10.1083/jcb.77.3.r27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Stacey D. W., Allfrey V. G. Evidence for the autophagy of microinjected proteins in HeLA cells. J Cell Biol. 1977 Dec;75(3):807–817. doi: 10.1083/jcb.75.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Taylor D. L., Wang Y. L. Fluorescently labelled molecules as probes of the structure and function of living cells. Nature. 1980 Apr 3;284(5755):405–410. doi: 10.1038/284405a0. [DOI] [PubMed] [Google Scholar]
  16. Ternynck T., Avrameas S. A new method using p-benzoquinone for coupling antigens and antibodies to marker substances. Ann Immunol (Paris) 1976 Mar-Apr;127(2):197–208. [PubMed] [Google Scholar]
  17. Weeds A. Actin-binding proteins--regulators of cell architecture and motility. Nature. 1982 Apr 29;296(5860):811–816. doi: 10.1038/296811a0. [DOI] [PubMed] [Google Scholar]
  18. Wehland J., Willingham M. C., Sandoval I. V. A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. I. Biochemical characterization, effects on microtubule polymerization in vitro, and microtubule polymerization and organization in vivo. J Cell Biol. 1983 Nov;97(5 Pt 1):1467–1475. doi: 10.1083/jcb.97.5.1467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Yin H. L., Stossel T. P. Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein. Nature. 1979 Oct 18;281(5732):583–586. doi: 10.1038/281583a0. [DOI] [PubMed] [Google Scholar]

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