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. 1991 Jul 1;114(1):73–81. doi: 10.1083/jcb.114.1.73

Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate

PMCID: PMC2289054  PMID: 2050742

Abstract

Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build- up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP-tubulin association.

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Selected References

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  1. Bayley P. M., Schilstra M. J., Martin S. R. Microtubule dynamic instability: numerical simulation of microtubule transition properties using a Lateral Cap model. J Cell Sci. 1990 Jan;95(Pt 1):33–48. doi: 10.1242/jcs.95.1.33. [DOI] [PubMed] [Google Scholar]
  2. Bell C. W., Fraser C., Sale W. S., Tang W. J., Gibbons I. R. Preparation and purification of dynein. Methods Cell Biol. 1982;24:373–397. doi: 10.1016/s0091-679x(08)60666-4. [DOI] [PubMed] [Google Scholar]
  3. Belmont L. D., Hyman A. A., Sawin K. E., Mitchison T. J. Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell. 1990 Aug 10;62(3):579–589. doi: 10.1016/0092-8674(90)90022-7. [DOI] [PubMed] [Google Scholar]
  4. Berg H. C., Block S. M. A miniature flow cell designed for rapid exchange of media under high-power microscope objectives. J Gen Microbiol. 1984 Nov;130(11):2915–2920. doi: 10.1099/00221287-130-11-2915. [DOI] [PubMed] [Google Scholar]
  5. Caplow M., Reid R. Directed elongation model for microtubule GTP hydrolysis. Proc Natl Acad Sci U S A. 1985 May;82(10):3267–3271. doi: 10.1073/pnas.82.10.3267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Caplow M., Ruhlen R., Shanks J., Walker R. A., Salmon E. D. Stabilization of microtubules by tubulin-GDP-Pi subunits. Biochemistry. 1989 Oct 3;28(20):8136–8141. doi: 10.1021/bi00446a026. [DOI] [PubMed] [Google Scholar]
  7. Caplow M., Shanks J., Ruhlen R. L. Temperature-jump studies of microtubule dynamic instability. J Biol Chem. 1988 Jul 25;263(21):10344–10352. [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Carlier M. F., Pantaloni D. Kinetic analysis of guanosine 5'-triphosphate hydrolysis associated with tubulin polymerization. Biochemistry. 1981 Mar 31;20(7):1918–1924. doi: 10.1021/bi00510a030. [DOI] [PubMed] [Google Scholar]
  11. Cassimeris L. U., Wadsworth P., Salmon E. D. Dynamics of microtubule depolymerization in monocytes. J Cell Biol. 1986 Jun;102(6):2023–2032. doi: 10.1083/jcb.102.6.2023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cassimeris L. U., Walker R. A., Pryer N. K., Salmon E. D. Dynamic instability of microtubules. Bioessays. 1987 Oct;7(4):149–154. doi: 10.1002/bies.950070403. [DOI] [PubMed] [Google Scholar]
  13. Cassimeris L., Pryer N. K., Salmon E. D. Real-time observations of microtubule dynamic instability in living cells. J Cell Biol. 1988 Dec;107(6 Pt 1):2223–2231. doi: 10.1083/jcb.107.6.2223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chen Y. D., Hill T. L. Monte Carlo study of the GTP cap in a five-start helix model of a microtubule. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1131–1135. doi: 10.1073/pnas.82.4.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. David-Pfeuty T., Erickson H. P., Pantaloni D. Guanosinetriphosphatase activity of tubulin associated with microtubule assembly. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5372–5376. doi: 10.1073/pnas.74.12.5372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hayden J. H., Bowser S. S., Rieder C. L. Kinetochores capture astral microtubules during chromosome attachment to the mitotic spindle: direct visualization in live newt lung cells. J Cell Biol. 1990 Sep;111(3):1039–1045. doi: 10.1083/jcb.111.3.1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hiller G., Weber K. Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues. Cell. 1978 Aug;14(4):795–804. doi: 10.1016/0092-8674(78)90335-5. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Kellogg D. R., Field C. M., Alberts B. M. Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo. J Cell Biol. 1989 Dec;109(6 Pt 1):2977–2991. doi: 10.1083/jcb.109.6.2977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kirschner M. W., Mitchison T. Microtubule dynamics. Nature. 1986 Dec 18;324(6098):621–621. doi: 10.1038/324621a0. [DOI] [PubMed] [Google Scholar]
  21. Kirschner M., Mitchison T. Beyond self-assembly: from microtubules to morphogenesis. Cell. 1986 May 9;45(3):329–342. doi: 10.1016/0092-8674(86)90318-1. [DOI] [PubMed] [Google Scholar]
  22. Kobayashi T. Dephosphorylation of tubulin-bound guanosine triphosphate during microtubule assembly. J Biochem. 1975 Jun;77(6):1193–1197. [PubMed] [Google Scholar]
  23. Leslie R. J., Saxton W. M., Mitchison T. J., Neighbors B., Salmon E. D., McIntosh J. R. Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching. J Cell Biol. 1984 Dec;99(6):2146–2156. doi: 10.1083/jcb.99.6.2146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Melki R., Carlier M. F., Pantaloni D. Direct evidence for GTP and GDP-Pi intermediates in microtubule assembly. Biochemistry. 1990 Sep 25;29(38):8921–8932. doi: 10.1021/bi00490a007. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. O'Brien E. T., Voter W. A., Erickson H. P. GTP hydrolysis during microtubule assembly. Biochemistry. 1987 Jun 30;26(13):4148–4156. doi: 10.1021/bi00387a061. [DOI] [PubMed] [Google Scholar]
  27. Pantaloni D., Carlier M. F. Involvement of guanosine triphosphate (GTP) hydrolysis in the mechanism of tubulin polymerization: regulation of microtubule dynamics at steady state by a GTP cap. Ann N Y Acad Sci. 1986;466:496–509. doi: 10.1111/j.1749-6632.1986.tb38427.x. [DOI] [PubMed] [Google Scholar]
  28. Rieder C. L., Alexander S. P. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells. J Cell Biol. 1990 Jan;110(1):81–95. doi: 10.1083/jcb.110.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rieder C. L., Davison E. A., Jensen L. C., Cassimeris L., Salmon E. D. Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half-spindle. J Cell Biol. 1986 Aug;103(2):581–591. doi: 10.1083/jcb.103.2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sammak P. J., Borisy G. G. Direct observation of microtubule dynamics in living cells. Nature. 1988 Apr 21;332(6166):724–726. doi: 10.1038/332724a0. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Schilstra M. J., Martin S. R., Bayley P. M. On the relationship between nucleotide hydrolysis and microtubule assembly: studies with a GTP-regenerating system. Biochem Biophys Res Commun. 1987 Sep 15;147(2):588–595. doi: 10.1016/0006-291x(87)90971-5. [DOI] [PubMed] [Google Scholar]
  34. Schulze E., Kirschner M. Microtubule dynamics in interphase cells. J Cell Biol. 1986 Mar;102(3):1020–1031. doi: 10.1083/jcb.102.3.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schulze E., Kirschner M. New features of microtubule behaviour observed in vivo. Nature. 1988 Jul 28;334(6180):356–359. doi: 10.1038/334356a0. [DOI] [PubMed] [Google Scholar]
  36. Sloboda R. D., Rosenbaum J. L. Purification and assay of microtubule-associated proteins (MAPs). Methods Enzymol. 1982;85(Pt B):409–416. doi: 10.1016/0076-6879(82)85041-6. [DOI] [PubMed] [Google Scholar]
  37. Stewart R. J., Farrell K. W., Wilson L. Role of GTP hydrolysis in microtubule polymerization: evidence for a coupled hydrolysis mechanism. Biochemistry. 1990 Jul 10;29(27):6489–6498. doi: 10.1021/bi00479a022. [DOI] [PubMed] [Google Scholar]
  38. Vallee R. B., Collins C. A. 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. doi: 10.1016/0076-6879(86)34080-1. [DOI] [PubMed] [Google Scholar]
  39. Verde F., Labbé J. C., Dorée M., Karsenti E. Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature. 1990 Jan 18;343(6255):233–238. doi: 10.1038/343233a0. [DOI] [PubMed] [Google Scholar]
  40. Voter W. A., Erickson H. P. The kinetics of microtubule assembly. Evidence for a two-stage nucleation mechanism. J Biol Chem. 1984 Aug 25;259(16):10430–10438. [PubMed] [Google Scholar]
  41. Voter W. A., O'Brien E. T., Erickson H. P. Dilution-induced disassembly of microtubules: relation to dynamic instability and the GTP cap. Cell Motil Cytoskeleton. 1991;18(1):55–62. doi: 10.1002/cm.970180106. [DOI] [PubMed] [Google Scholar]
  42. Wadsworth P., Salmon E. D. Microtubule dynamics in mitotic spindles of living cells. Ann N Y Acad Sci. 1986;466:580–592. doi: 10.1111/j.1749-6632.1986.tb38434.x. [DOI] [PubMed] [Google Scholar]
  43. Wadsworth P., Salmon E. D. Preparation and characterization of fluorescent analogs of tubulin. Methods Enzymol. 1986;134:519–528. doi: 10.1016/0076-6879(86)34117-x. [DOI] [PubMed] [Google Scholar]
  44. Walker R. A., Inoué S., Salmon E. D. Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro. J Cell Biol. 1989 Mar;108(3):931–937. doi: 10.1083/jcb.108.3.931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Walker R. A., O'Brien E. T., Pryer N. K., Soboeiro M. F., Voter W. A., Erickson H. P., Salmon E. D. Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies. J Cell Biol. 1988 Oct;107(4):1437–1448. doi: 10.1083/jcb.107.4.1437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Weisenberg R. C., Deery W. J., Dickinson P. J. Tubulin-nucleotide interactions during the polymerization and depolymerization of microtubules. Biochemistry. 1976 Sep 21;15(19):4248–4254. doi: 10.1021/bi00664a018. [DOI] [PubMed] [Google Scholar]

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