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. 1986 Feb 1;102(2):619–627. doi: 10.1083/jcb.102.2.619

End-to-end annealing of microtubules in vitro

PMCID: PMC2114097  PMID: 3511075

Abstract

Mixtures of pre-formed microtubules, polymerized from chicken erythrocyte and brain tubulin, rapidly anneal end-to-end in vitro in standard microtubule assembly buffer. The erythrocyte tubulin segments in annealed heteropolymers can be distinguished by an immunoelectron microscopic assay that uses an antibody specific for chicken erythrocyte beta-tubulin. An annealing process is consistent with the following observations: (a) Microtubule number decreases while the polymer mass remains constant. (b) As the total number of microtubules declines, the number of heteropolymers, and the number of segments contained in each heteropolymer, increases. (c) The size of the segments determined after annealing and antibody labeling is the same as the original microtubule polymers. (d) Points of discontinuity in the annealing heteropolymers can be observed directly by electron microscopy, and correspond to type-specific polymer domains. The junctions probably represent initial contact points during the annealing process. Microtubule annealing occurs rapidly in vitro and may be significant for determining properties of microtubule dynamics in vivo.

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Selected References

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

  1. Behnke O. Microtubules in disk-shaped blood cells. Int Rev Exp Pathol. 1970;9:1–92. [PubMed] [Google Scholar]
  2. Bradford M. M. 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. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Carlier M. F., Hill T. L., Chen Y. Interference of GTP hydrolysis in the mechanism of microtubule assembly: an experimental study. Proc Natl Acad Sci U S A. 1984 Feb;81(3):771–775. doi: 10.1073/pnas.81.3.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carlier M. F., Pantaloni D., Korn E. D. Steady state length distribution of F-actin under controlled fragmentation and mechanism of length redistribution following fragmentation. J Biol Chem. 1984 Aug 25;259(16):9987–9991. [PubMed] [Google Scholar]
  5. Carlier M. F., Pantaloni D. Taxol effect on tubulin polymerization and associated guanosine 5'-triphosphate hydrolysis. Biochemistry. 1983 Sep 27;22(20):4814–4822. doi: 10.1021/bi00289a031. [DOI] [PubMed] [Google Scholar]
  6. Cooper J. A., Buhle E. L., Jr, Walker S. B., Tsong T. Y., Pollard T. D. Kinetic evidence for a monomer activation step in actin polymerization. Biochemistry. 1983 Apr 26;22(9):2193–2202. doi: 10.1021/bi00278a021. [DOI] [PubMed] [Google Scholar]
  7. Dentler W. L., Granett S., Rosenbaum J. L. Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules. J Cell Biol. 1975 Apr;65(1):237–241. doi: 10.1083/jcb.65.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Euteneuer U., Ris H., Borisy G. G. Polarity of marginal-band microtubules in vertebrate erythrocytes. Eur J Cell Biol. 1985 May;37:149–155. [PubMed] [Google Scholar]
  9. Frieden C., Goddette D. W. Polymerization of actin and actin-like systems: evaluation of the time course of polymerization in relation to the mechanism. Biochemistry. 1983 Dec 6;22(25):5836–5843. doi: 10.1021/bi00294a023. [DOI] [PubMed] [Google Scholar]
  10. Gottlieb R. A., Murphy D. B. The pattern of MAP-2 binding on microtubules: visual enhancement of MAP attachment sites by antibody labeling and electron microscopy. J Ultrastruct Res. 1983 Nov;85(2):175–185. doi: 10.1016/s0022-5320(83)90105-3. [DOI] [PubMed] [Google Scholar]
  11. Hill T. L., Carlier M. F. Steady-state theory of the interference of GTP hydrolysis in the mechanism of microtubule assembly. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7234–7238. doi: 10.1073/pnas.80.23.7234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hill T. L. Introductory analysis of the GTP-cap phase-change kinetics at the end of a microtubule. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6728–6732. doi: 10.1073/pnas.81.21.6728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Johnson K. A., Borisy G. G. Kinetic analysis of microtubule self-assembly in vitro. J Mol Biol. 1977 Nov 25;117(1):1–31. doi: 10.1016/0022-2836(77)90020-1. [DOI] [PubMed] [Google Scholar]
  14. Kawamura M., Maruyama K. A further study of electron microscopic particle length of F-actin polymerized in vitro. J Biochem. 1972 Jul;72(1):179–188. doi: 10.1093/oxfordjournals.jbchem.a129884. [DOI] [PubMed] [Google Scholar]
  15. Kondo H., Ishiwata S. Uni-directional growth of F-actin. J Biochem. 1976 Jan;79(1):159–171. doi: 10.1093/oxfordjournals.jbchem.a131043. [DOI] [PubMed] [Google Scholar]
  16. Kristofferson D., Karr T. L., Purich D. L. Dynamics of linear protein polymer disassembly. J Biol Chem. 1980 Sep 25;255(18):8567–8572. [PubMed] [Google Scholar]
  17. MacNeal R. K., Webb B. C., Purich D. L. Neurotubule assembly at substoichiometric nucleotide levels using a GTP regenerating system. Biochem Biophys Res Commun. 1977 Jan 24;74(2):440–447. doi: 10.1016/0006-291x(77)90323-0. [DOI] [PubMed] [Google Scholar]
  18. Margolis R. L., Wilson L. Microtubule treadmills--possible molecular machinery. Nature. 1981 Oct 29;293(5835):705–711. doi: 10.1038/293705a0. [DOI] [PubMed] [Google Scholar]
  19. Mitchison T., Kirschner M. Dynamic instability of microtubule growth. Nature. 1984 Nov 15;312(5991):237–242. doi: 10.1038/312237a0. [DOI] [PubMed] [Google Scholar]
  20. Mitchison T., Kirschner M. Microtubule assembly nucleated by isolated centrosomes. Nature. 1984 Nov 15;312(5991):232–237. doi: 10.1038/312232a0. [DOI] [PubMed] [Google Scholar]
  21. Murphy D. B., Wallis K. T. Brain and erythrocyte microtubules from chicken contain different beta-tubulin polypeptides. J Biol Chem. 1983 Jun 25;258(12):7870–7875. [PubMed] [Google Scholar]
  22. Murphy D. B., Wallis K. T. Erythrocyte microtubule assembly in vitro. Determination of the effects of erythrocyte tau, tubulin isoforms, and tubulin oligomers on erythrocyte tubulin assembly, and comparison with brain microtubule assembly. J Biol Chem. 1985 Oct 5;260(22):12293–12301. [PubMed] [Google Scholar]
  23. Murphy D. B., Wallis K. T. Isolation of microtubule protein from chicken erythrocytes and determination of the critical concentration for tubulin polymerization in vitro and in vivo. J Biol Chem. 1983 Jul 10;258(13):8357–8364. [PubMed] [Google Scholar]
  24. Nachmias V. T. Cytoskeleton of human platelets at rest and after spreading. J Cell Biol. 1980 Sep;86(3):795–802. doi: 10.1083/jcb.86.3.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nakaoka Y., Kasai M. Behaviour of sonicated actin polymers: adenosine triphosphate splitting and polymerization. J Mol Biol. 1969 Sep 14;44(2):319–332. doi: 10.1016/0022-2836(69)90178-8. [DOI] [PubMed] [Google Scholar]
  26. Roobol A., Pogson C. I., Gull K. Identification and characterization of microtubule proteins from myxamoebae of Physarum polycephalum. Biochem J. 1980 Aug 1;189(2):305–312. doi: 10.1042/bj1890305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rothwell S. W., Grasser W. A., Murphy D. B. Direct observation of microtubule treadmilling by electron microscopy. J Cell Biol. 1985 Nov;101(5 Pt 1):1637–1642. doi: 10.1083/jcb.101.5.1637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Saxton W. M., Stemple D. L., Leslie R. J., Salmon E. D., Zavortink M., McIntosh J. R. Tubulin dynamics in cultured mammalian cells. J Cell Biol. 1984 Dec;99(6):2175–2186. doi: 10.1083/jcb.99.6.2175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Slot J. W., Geuze H. J. Sizing of protein A-colloidal gold probes for immunoelectron microscopy. J Cell Biol. 1981 Aug;90(2):533–536. doi: 10.1083/jcb.90.2.533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wegner A., Savko P. Fragmentation of actin filaments. Biochemistry. 1982 Apr 13;21(8):1909–1913. doi: 10.1021/bi00537a032. [DOI] [PubMed] [Google Scholar]

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