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J Cell Biol. Oct 1, 1988; 107(4): 1437–1448.
PMCID: PMC2115242

Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies

Abstract

We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37 degrees C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 microM. Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 microM-1 s-1 for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s-1. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 microM. The rate of rapid shortening was similar at the two ends (plus = 733 s-1; minus = 915 s-1), and did not vary with tubulin concentration. Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Allen RD, Allen NS, Travis JL. Video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: a new method capable of analyzing microtubule-related motility in the reticulopodial network of Allogromia laticollaris. Cell Motil. 1981;1(3):291–302. [PubMed]
  • Bell CW, Fraser C, Sale WS, Tang WJ, Gibbons IR. Preparation and purification of dynein. Methods Cell Biol. 1982;24:373–397. [PubMed]
  • Bergen LG, Borisy GG. Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly. J Cell Biol. 1980 Jan;84(1):141–150. [PMC free article] [PubMed]
  • Borisy GG, Bergen LG. A direct method for analyzing the polymerization kinetics at the two ends of a microtubule. Methods Cell Biol. 1982;24:171–187. [PubMed]
  • Caplow M. Location of the guanosine triphosphate (GTP) hydrolysis site in microtubules. Ann N Y Acad Sci. 1986;466:510–518. [PubMed]
  • Caplow M, Reid R. Directed elongation model for microtubule GTP hydrolysis. Proc Natl Acad Sci U S A. 1985 May;82(10):3267–3271. [PMC free article] [PubMed]
  • Carlier MF, Pantaloni D. Kinetic analysis of guanosine 5'-triphosphate hydrolysis associated with tubulin polymerization. Biochemistry. 1981 Mar 31;20(7):1918–1924. [PubMed]
  • Carlier MF, 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. [PubMed]
  • Carlier MF, Hill TL, 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. [PMC free article] [PubMed]
  • Cassimeris LU, Wadsworth P, Salmon ED. Dynamics of microtubule depolymerization in monocytes. J Cell Biol. 1986 Jun;102(6):2023–2032. [PMC free article] [PubMed]
  • Cassimeris LU, Walker RA, Pryer NK, Salmon ED. Dynamic instability of microtubules. Bioessays. 1987 Oct;7(4):149–154. [PubMed]
  • Chen YD, Hill TL. 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. [PMC free article] [PubMed]
  • David-Pfeuty T, Erickson HP, Pantaloni D. Guanosinetriphosphatase activity of tubulin associated with microtubule assembly. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5372–5376. [PMC free article] [PubMed]
  • Farrell KW, Jordan MA, Miller HP, Wilson L. Phase dynamics at microtubule ends: the coexistence of microtubule length changes and treadmilling. J Cell Biol. 1987 Apr;104(4):1035–1046. [PMC free article] [PubMed]
  • Gard DL, Kirschner MW. A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end. J Cell Biol. 1987 Nov;105(5):2203–2215. [PMC free article] [PubMed]
  • Hill TL, 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. [PMC free article] [PubMed]
  • Horio T, Hotani H. Visualization of the dynamic instability of individual microtubules by dark-field microscopy. Nature. 1986 Jun 5;321(6070):605–607. [PubMed]
  • Johnson KA, Borisy GG. Kinetic analysis of microtubule self-assembly in vitro. J Mol Biol. 1977 Nov 25;117(1):1–31. [PubMed]
  • Karr TL, Podrasky AE, Purich DL. Participation of guanine nucleotides in nucleation and elongation steps of microtubule assembly. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5475–5479. [PMC free article] [PubMed]
  • Kirschner MW, Mitchison T. Microtubule dynamics. Nature. 1986 Dec 18;324(6098):621–621. [PubMed]
  • Kobayashi T. Dephosphorylation of tubulin-bound guanosine triphosphate during microtubule assembly. J Biochem. 1975 Jun;77(6):1193–1197. [PubMed]
  • Kristofferson D, Mitchison T, Kirschner M. Direct observation of steady-state microtubule dynamics. J Cell Biol. 1986 Mar;102(3):1007–1019. [PMC free article] [PubMed]
  • Leslie RJ, Saxton WM, Mitchison TJ, Neighbors B, Salmon ED, McIntosh JR. Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching. J Cell Biol. 1984 Dec;99(6):2146–2156. [PMC free article] [PubMed]
  • Lutz DA, Inoué S. Techniques for observing living gametes and embryos. Methods Cell Biol. 1986;27:89–110. [PubMed]
  • Merril CR, Goldman D, Sedman SA, Ebert MH. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. [PubMed]
  • Mitchison T, Kirschner M. Microtubule assembly nucleated by isolated centrosomes. Nature. 1984 Nov 15;312(5991):232–237. [PubMed]
  • Mitchison T, Kirschner M. Dynamic instability of microtubule growth. Nature. 1984 Nov 15;312(5991):237–242. [PubMed]
  • Mitchison T, Evans L, Schulze E, Kirschner M. Sites of microtubule assembly and disassembly in the mitotic spindle. Cell. 1986 May 23;45(4):515–527. [PubMed]
  • Morrissey JH. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. [PubMed]
  • O'Brien ET, Voter WA, Erickson HP. GTP hydrolysis during microtubule assembly. Biochemistry. 1987 Jun 30;26(13):4148–4156. [PubMed]
  • Penningroth SM, Kirschner MW. Nucleotide binding and phosphorylation in microtubule assembly in vitro. J Mol Biol. 1977 Oct 5;115(4):643–673. [PubMed]
  • Pryer NK, Wadsworth P, Salmon ED. Polarized microtubule gliding and particle saltations produced by soluble factors from sea urchin eggs and embryos. Cell Motil Cytoskeleton. 1986;6(6):537–548. [PubMed]
  • Sammak PJ, Gorbsky GJ, Borisy GG. Microtubule dynamics in vivo: a test of mechanisms of turnover. J Cell Biol. 1987 Mar;104(3):395–405. [PMC free article] [PubMed]
  • Schnapp BJ. Viewing single microtubules by video light microscopy. Methods Enzymol. 1986;134:561–573. [PubMed]
  • Schulze E, Kirschner M. Microtubule dynamics in interphase cells. J Cell Biol. 1986 Mar;102(3):1020–1031. [PMC free article] [PubMed]
  • Voter WA, Erickson HP. Electron microscopy of MAP 2 (microtubule-associated protein 2). J Ultrastruct Res. 1982 Sep;80(3):374–382. [PubMed]
  • Voter WA, Erickson HP. The kinetics of microtubule assembly. Evidence for a two-stage nucleation mechanism. J Biol Chem. 1984 Aug 25;259(16):10430–10438. [PubMed]
  • Wadsworth P, Salmon ED. Analysis of the treadmilling model during metaphase of mitosis using fluorescence redistribution after photobleaching. J Cell Biol. 1986 Mar;102(3):1032–1038. [PMC free article] [PubMed]
  • Wadsworth P, Salmon ED. Preparation and characterization of fluorescent analogs of tubulin. Methods Enzymol. 1986;134:519–528. [PubMed]
  • Wegner A. Head to tail polymerization of actin. J Mol Biol. 1976 Nov;108(1):139–150. [PubMed]
  • Weisenberg RC, Deery WJ, Dickinson PJ. Tubulin-nucleotide interactions during the polymerization and depolymerization of microtubules. Biochemistry. 1976 Sep 21;15(19):4248–4254. [PubMed]
  • Yanagisawa T, Hasegawa S, Mohri H. The bound nucleotides of the isolated microtubules of sea-urchin sperm flagella and their possible role in flagellar movement. Exp Cell Res. 1968 Sep;52(1):86–100. [PubMed]
  • Zeeberg B, Caplow M. An isoenergetic exchange mechanism which accounts for tubulin-GDP stabilization of microtubules. J Biol Chem. 1981 Dec 10;256(23):12051–12057. [PubMed]

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