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Cellular and Molecular Bioengineering

, Volume 6, Issue 4, pp 383–392 | Cite as

Macromolecular Crowding Pushes Catalyzed Microtubule Growth to Near the Theoretical Limit

  • Michal Wieczorek
  • Sami Chaaban
  • Gary J. Brouhard
Article

Abstract

Microtubule growth is accelerated by enzymes such as XMAP215, but in vivo microtubule assembly rates remain much higher than in vitro reconstitution assays using only purified components. Recently, XMAP215 and EB1 have been shown to synergistically enhance microtubule growth to near physiological rates. The growth rates reported remain lower, however, than those observed in C. elegans embryos and the theoretical upper limit derived from mass-transfer models. It is possible that the crowded environment of the cytoplasm creates an “excluded volume” effect, which typically accelerates biochemical reactions and could account for this discrepancy. We sought to determine the effects of macromolecular crowding agents on microtubule growth rates. We found that the apparent rate constant for tubulin addition increased up to 10-fold in viscous environments with large macromolecules. In contrast, increasing the viscosity with small solutes decreased growth rates in a manner consistent with tubulin binding to microtubule ends in a diffusion-limited reaction. Adding crowding agents with XMAP215 and EB1 resulted in growth rates that saturated at ∼45 μm/min at 10 μM tubulin. To our knowledge, this represents the fastest in vitro microtubule growth rates measured to date and approaches the theoretical limit.

Keywords

Microtubule Tubulin Polymerization XMAP215 EB1 Diffusion Excluded volume Macromolecular crowding Isomerization 

Notes

Acknowledgments

This paper is dedicated to Alan Hunt (1963–2012), who served as Ph.D. supervisor to G.J.B. Alan’s energy, intelligence and dedication to science were an inspiration. We are grateful to Dr. Elizabeth Jones for the use of her microrheometer. We thank Abattoir Jacques Forget (Terrebonne, Québec) for source material for tubulin purification. We thank S. Bechstedt for her broad support of the molecular biology and protein purification that underly this work. This work was supported by operating grants from the Canadian Institutes of Health Research (CIHR MOP-111265) and the Natural Sciences and Engineering Research Council (NSERC #372593-09). M.W. is supported by an NSERC Canada Graduate Scholarship. S.C. is supported by an NSERC scholarship through the Cellular Dynamics of Macromolecular Complexes training program. G.J.B. is the recipient of a CIHR New Investigator Award.

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References

  1. 1.
    Adames, N. R., and J. A. Cooper. Microtubule interactions with the cell cortex causing nuclear movements in saccharomyces cerevisiae. J. Cell Biol. 149(4):863–874, 03Google Scholar
  2. 2.
    Ayaz, P., X. Ye, P. Huddleston, C. A. Brautigam, and L. M. Rice. A TOG:\(\alpha/\beta\)-tubulin complex structure reveals conformation-based mechanisms for a microtubule polymerase. Science 337(6096):857–860, 2012.CrossRefGoogle Scholar
  3. 3.
    Bechstedt, S., and G. J. Brouhard. Doublecortin recognizes the 13-protofilament microtubule cooperatively and tracks microtubule ends. Dev. Cell 23(1):181–192, 2012.CrossRefGoogle Scholar
  4. 4.
    Belmont, L. D., A. A. Hyman, K. E. Sawin, and T. J. Mitchison. Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 62:579–589, 1990.CrossRefGoogle Scholar
  5. 5.
    Berg, O. G., and P. H. von Hippel. Diffusion-controlled macromolecular interactions. Annu. Rev. Biophys. Biophys. Chem. 14:131–160, 1985.CrossRefGoogle Scholar
  6. 6.
    Brouhard, G. J., J. H. Stear, T. L. Noetzel, J. Al-Bassam, K. Kinoshita, S. C. Harrison, J. Howard, and A. A. Hyman. XMAP 215 is a processive microtubule polymerase. Cell 132(1):79–88, 2008.Google Scholar
  7. 7.
    Castoldi, M., and A. V. Popov. Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr. Purif. 32(1):83–88, 2003.CrossRefGoogle Scholar
  8. 8.
    Compton, D. A. Spindle assembly in animal cells. Annu. Rev. Biochem. 69:95–114, 2000.CrossRefGoogle Scholar
  9. 9.
    Dent, E. W., and F. B. Gertler. Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40(2):209–227, 2003.CrossRefGoogle Scholar
  10. 10.
    Drechsel, D. N., A. A. Hyman, M. H. Cobb, and M. W. Kirschner. Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau. Mol. Biol. Cell 3(10):1141–1154, 1992.CrossRefGoogle Scholar
  11. 11.
    Drenckhahn, D., and T. D. Pollard. Elongation of actin filaments is a diffusion-limited reaction at the barbed end and is accelerated by inert macromolecules. J. Biol. Chem. 261(27):12754–12758, 1986.Google Scholar
  12. 12.
    Gard, D. L., and M. W. Kirschner. A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end. J. Cell Biol. 105(5):2203–2215, 1987.CrossRefGoogle Scholar
  13. 13.
    Gardner, M. K., B. D. Charlebois, I. M. Janosi, J. Howard, A. J. Hunt, and D. J. Odde. Rapid microtubule self-assembly kinetics. Cell 146(4):582–592, 2011.CrossRefGoogle Scholar
  14. 14.
    Gell, C., V. Bormuth, G. J. Brouhard, D. N. Cohen, S. Diez, C. T. Friel, J. Helenius, B. Nitzsche, H. Petzold, J. Ribbe, E. Schaffer, J. H. Stear, A. Trushko, V. Varga, P. O. Widlund, M. Zanic, and J. Howard. Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy. Methods Cell Biol. 95:221–245, 2010.Google Scholar
  15. 15.
    Glotzer, M. The 3Ms of central spindle assembly: microtubules, motors and MAPs. Nat. Rev. Mol. Cell Biol. 10(1):9–20, 2009.CrossRefGoogle Scholar
  16. 16.
    Helenius, J., G. Brouhard, Y. Kalaidzidis, S. Diez, and J. Howard. The depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtubule ends. Nature 441(7089):115–119, 2006.CrossRefGoogle Scholar
  17. 17.
    Hiller, G., and K. Weber. Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues. Cell 14(4):795–804, 1978.CrossRefGoogle Scholar
  18. 18.
    Honnappa, S., S. M. Gouveia, A. Weisbrich, F. F. Damberger, N. S. Bhavesh, H. Jawhari, I. Grigoriev, F. J. A. van Rijssel, R. M. Buey, A. Lawera, I. Jelesarov, F. K. Winkler, K. Wuthrich, A. Akhmanova, and M. O. Steinmetz. An EB1-binding motif acts as a microtubule tip localization signal. Cell 138(2):366–376, 2009.Google Scholar
  19. 19.
    Hyman, A., D. Drechsel, D. Kellogg, S. Salser, K. Sawin, P. Steffen, L. Wordeman, and T. Mitchison. Preparation of modified tubulins. Methods Enzymol. 196:478–485, 1991.CrossRefGoogle Scholar
  20. 20.
    Kinoshita, K., B. Habermann, and A. A. Hyman. XMAP215: a key component of the dynamic microtubule cytoskeleton. Trends Cell Biol. 12(6):267–273, 2002.CrossRefGoogle Scholar
  21. 21.
    Kuchnir Fygenson, D., H. Flyvbjerg, K. Sneppen, A. Libchaber, and S. Leibler. Spontaneous nucleation of microtubules. Phys. Rev. E 51(5):5058–5063, 1995.CrossRefGoogle Scholar
  22. 22.
    McGuffee, S. R., and A. H. Elcock. Diffusion, crowding and protein stability in a dynamic molecular model of the bacterial cytoplasm. PLoS Comput. Biol. 6(3):e1000694, 03, 2010.Google Scholar
  23. 23.
    Minton, A. P. Excluded volume as a determinant of macromolecular structure and reactivity. Biopolymers 20(10):2093–2120, 1981.CrossRefGoogle Scholar
  24. 24.
    Morrison, E. E., B. N. Wardleworth, J. M. Askham, A. F. Markham, and D. M. Meredith. EB1, a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle. Oncogene 17(26):3471–3477, 2000.Google Scholar
  25. 25.
    Nogales, E., S. G. Wolf, and K. H. Downing. Structure of the \(\alpha/\beta\)-tubulin dimer by electron crystallography. Nature 391(6663):199–203, 1998.Google Scholar
  26. 26.
    Norholm, M. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10(1):21, 2010.CrossRefGoogle Scholar
  27. 27.
    Northrup, S. H., and H. P. Erickson. Kinetics of protein-protein association explained by brownian dynamics computer simulation. Proc. Natl Acad. Sci. USA. 89(8):3338–3342, 1992.CrossRefGoogle Scholar
  28. 28.
    Odde, D. J. Estimation of the diffusion-limited rate of microtubule assembly. Biophys. J. 73(1):88–96, 1997.CrossRefGoogle Scholar
  29. 29.
    Oosawa, F., and S. Asakura. Thermodynamics of the Polymerization of Protein, Vol. 20. London: Academic Press, 1975.Google Scholar
  30. 30.
    Pecqueur, L., C. Duellberg, B. Dreier, Q. Jiang, C. Wang, A. Pluckthun, T. Surrey, B. Gigant, and M. Knossow. A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end. Proc. Natl Acad. Sci. USA. 109(30):12011–12016, 2000.Google Scholar
  31. 31.
    Ravelli, R. B., B. Gigant, P. A. Curmi, I. Jourdain, S. Lachkar, A. Sobel, and M. Knossow. Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature 428(6979):198–202, 2004.CrossRefGoogle Scholar
  32. 32.
    Rice, L. M., E. A. Montabana, and D. A. Agard. The lattice as allosteric effector: structural studies of α/β- and γ-tubulin clarify the role of GTP in microtubule assembly. Proc. Natl Acad. Sci. USA. 105(14):5378–5383, 2008.CrossRefGoogle Scholar
  33. 33.
    Rogers, K. R., S. Weiss, I. Crevel, P. J. Brophy, M. Geeves, and R. Cross. KIF1D is a fast non-processive kinesin that demonstrates novel K-loop-dependent mechanochemistry. EMBO J. 20(18):5101–5113, 2001.Google Scholar
  34. 34.
    Salmon, E. D., W. M. Saxton, R. J. Leslie, M. L. Karow, and J. R. McIntosh. Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching. J. Cell Biol. 99(6):2157–2164, 1984.Google Scholar
  35. 35.
    Seksek, O., J. Biwersi, and A. S. Verkman. Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus. J. Cell Biol. 138(1):131–142, 1997.CrossRefGoogle Scholar
  36. 36.
    Shelanski, M. L., F. Gaskin, and C. R. Cantor. Microtubule assembly in the absence of added nucleotides. Proc. Natl Acad. Sci. USA. 70(3):765–768, 03, 1973.Google Scholar
  37. 37.
    Shelden, E., and P. Wadsworth. Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific. J. Cell Biol. 120(4):935–945, 1993.CrossRefGoogle Scholar
  38. 38.
    Srayko, M., A. Kaya, J. Stamford, and A. A. Hyman. Identification and characterization of factors required for microtubule growth and nucleation in the early C. elegans embryo. Dev. Cell 9(2):223–236, 2005.CrossRefGoogle Scholar
  39. 39.
    Su, L. K., M. Burrell, D. E. Hill, J. Gyuris, R. Brent, R. Wiltshire, J. Trent, B. Vogelstein, and K. W. Kinzler APC binds to the novel protein EB1. Cancer Res. 55(14):2972–2977, 1995.Google Scholar
  40. 40.
    Varga, V., C. Leduc, V. Bormuth, S. Diez, and J. Howard. Kinesin-8 motors act cooperatively to mediate length-dependent microtubule depolymerization. Cell 138(6):1174–1183, 2009.CrossRefGoogle Scholar
  41. 41.
    Walker, R. A., E. T. O’Brien, N. K. Pryer, M. F. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies. J. Cell Biol. 107(4): 1437–1448, 1988.Google Scholar
  42. 42.
    Wang, H. W., and E. Nogales. Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly. Nature 435(7044):911–915, 2005.CrossRefGoogle Scholar
  43. 43.
    Waterman-Storer, C. M., R. A. Worthylake, B. P. Liu, K. Burridge, and E. D. Salmon. Microtubule growth activates Rac1 to promote lamellipodial protrusion in fibroblasts. Nat. Cell Biol. 1(1):45–50, 1999.CrossRefGoogle Scholar
  44. 44.
    Winey, M., and K. Bloom. Mitotic spindle form and function. Genetics 190(4):1197–1224, 2012.CrossRefGoogle Scholar
  45. 45.
    Yaffe, M. P., N. Stuurman, and R. D. Vale. Mitochondrial positioning in fission yeast is driven by association with dynamic microtubules and mitotic spindle poles. Proc. Natl Acad. Sci. USA. 100(20):11424–11428, 2003.CrossRefGoogle Scholar
  46. 46.
    Zanic, M., P. O. Widlund, A. A. Hyman, J. Howard. Synergy between XMAP 215 and EB1 increases microtubule growth rates to physiological levels. Nat. Cell Biol. 15(6):688–693, 2013.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2013

Authors and Affiliations

  • Michal Wieczorek
    • 1
  • Sami Chaaban
    • 1
  • Gary J. Brouhard
    • 1
  1. 1.Department of BiologyMcGill UniversityMontréalCanada

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