Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1988 Sep;7(9):2653–2659. doi: 10.1002/j.1460-2075.1988.tb03118.x

Oscillations in microtubule polymerization: the rate of GTP regeneration on tubulin controls the period.

R Melki 1, M F Carlier 1, D Pantaloni 1
PMCID: PMC457053  PMID: 3181136

Abstract

The essential reactions involved in the oscillatory kinetics of microtubule polymerization have been investigated. The rate of GDP dissociation from tubulin decreased cooperatively upon increasing tubulin concentration above 20 microM, consistent with the formation of GDP oligomers whose dissociation is rate limiting in nucleotide exchange. The apparent rate constant for nucleotide exchange at high tubulin concentration was 0.02 s-1 at 37 degrees C, which is the exact value needed in previous theoretical simulations to obtain oscillations with the real period of 70-80 s. A glass filter assay separating microtubules from oligomers and tubulin allowed nucleotide bound to non-microtubular tubulin during the oscillations to be monitored. In agreement with nucleotide exchange data, tubulin-bound GDP was found to oscillate in antiphase with microtubules. By varying the concentration of an enzymatic GTP-regenerating system, we could demonstrate that the period of the oscillations is directly controlled by the rate at which GTP is regenerated on tubulin, and oscillations can be observed under conditions where the dissociation of oligomers is no longer rate limiting. The possible physiological significance of GTP-regenerating systems in establishing synchrony in a microtubule population is evoked. The present data confirm and extend the model that we previously proposed to account for the oscillations.

Full text

PDF
2653

Images in this article

2657Image
on p.2657

Selected References

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

  1. Bigay J., Deterre P., Pfister C., Chabre M. Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP. EMBO J. 1987 Oct;6(10):2907–2913. doi: 10.1002/j.1460-2075.1987.tb02594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brylawski B. P., Caplow M. Rate for nucleotide release from tubulin. J Biol Chem. 1983 Jan 25;258(2):760–763. [PubMed] [Google Scholar]
  3. Carlier M. F., Didry D., Melki R., Chabre M., Pantaloni D. Stabilization of microtubules by inorganic phosphate and its structural analogues, the fluoride complexes of aluminum and beryllium. Biochemistry. 1988 May 17;27(10):3555–3559. doi: 10.1021/bi00410a005. [DOI] [PubMed] [Google Scholar]
  4. Carlier M. F., Didry D., Pantaloni D. Microtubule elongation and guanosine 5'-triphosphate hydrolysis. Role of guanine nucleotides in microtubule dynamics. Biochemistry. 1987 Jul 14;26(14):4428–4437. doi: 10.1021/bi00388a036. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Carlier M. F., Melki R., Pantaloni D., Hill T. L., Chen Y. Synchronous oscillations in microtubule polymerization. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5257–5261. doi: 10.1073/pnas.84.15.5257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carlier M. F., Pantaloni D. Binding of phosphate to F-ADP-actin and role of F-ADP-Pi-actin in ATP-actin polymerization. J Biol Chem. 1988 Jan 15;263(2):817–825. [PubMed] [Google Scholar]
  8. Cashel M., Lazzarini R. A., Kalbacher B. An improved method for thin-layer chromatography of nucleotide mixtures containing 32P-labelled orthophosphate. J Chromatogr. 1969 Mar 11;40(1):103–109. doi: 10.1016/s0021-9673(01)96624-5. [DOI] [PubMed] [Google Scholar]
  9. Chen Y. D., Hill T. L. Theoretical studies on oscillations in microtubule polymerization. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8419–8423. doi: 10.1073/pnas.84.23.8419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen Y., Hill T. L. Theoretical treatment of microtubules disappearing in solution. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4127–4131. doi: 10.1073/pnas.82.12.4127. [DOI] [PMC free article] [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., Chen Y. Phase changes at the end of a microtubule with a GTP cap. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5772–5776. doi: 10.1073/pnas.81.18.5772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Horio T., Hotani H. Visualization of the dynamic instability of individual microtubules by dark-field microscopy. Nature. 1986 Jun 5;321(6070):605–607. doi: 10.1038/321605a0. [DOI] [PubMed] [Google Scholar]
  14. Howard W. D., Timasheff S. N. GDP state of tubulin: stabilization of double rings. Biochemistry. 1986 Dec 16;25(25):8292–8300. doi: 10.1021/bi00373a025. [DOI] [PubMed] [Google Scholar]
  15. Huitorel P., Simon C., Pantaloni D. Nucleoside diphosphate kinase from brain. Purification and effect on microtubule assembly in vitro. Eur J Biochem. 1984 Oct 15;144(2):233–241. doi: 10.1111/j.1432-1033.1984.tb08455.x. [DOI] [PubMed] [Google Scholar]
  16. Jacobs M., Huitorel P. Tubulin-associated nucleoside diphosphokinase. Eur J Biochem. 1979 Sep;99(3):613–622. doi: 10.1111/j.1432-1033.1979.tb13294.x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Krebs H. A., Wiggins D. Phosphorylation of adenosine monophosphate in the mitochondrial matrix. Biochem J. 1978 Jul 15;174(1):297–301. doi: 10.1042/bj1740297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Mandelkow E. M., Lange G., Jagla A., Spann U., Mandelkow E. Dynamics of the microtubule oscillator: role of nucleotides and tubulin-MAP interactions. EMBO J. 1988 Feb;7(2):357–365. doi: 10.1002/j.1460-2075.1988.tb02821.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pantaloni D., Carlier M. F., Simon C., Batelier G. Mechanism of tubulin assembly: role of rings in the nucleation process and of associated proteins in the stabilization of microtubules. Biochemistry. 1981 Aug 4;20(16):4709–4716. doi: 10.1021/bi00519a029. [DOI] [PubMed] [Google Scholar]
  23. Pirollet F., Job D., Margolis R. L., Garel J. R. An oscillatory mode for microtubule assembly. EMBO J. 1987 Nov;6(11):3247–3252. doi: 10.1002/j.1460-2075.1987.tb02642.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sammak P. J., Gorbsky G. J., Borisy G. G. Microtubule dynamics in vivo: a test of mechanisms of turnover. J Cell Biol. 1987 Mar;104(3):395–405. doi: 10.1083/jcb.104.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schulze E., Kirschner M. Dynamic and stable populations of microtubules in cells. J Cell Biol. 1987 Feb;104(2):277–288. doi: 10.1083/jcb.104.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES