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.
This is a preview of subscription content, log in to check access.
Buy single article
Instant unlimited access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
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, 03
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.
Bechstedt, S., and G. J. Brouhard. Doublecortin recognizes the 13-protofilament microtubule cooperatively and tracks microtubule ends. Dev. Cell 23(1):181–192, 2012.
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.
Berg, O. G., and P. H. von Hippel. Diffusion-controlled macromolecular interactions. Annu. Rev. Biophys. Biophys. Chem. 14:131–160, 1985.
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.
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.
Compton, D. A. Spindle assembly in animal cells. Annu. Rev. Biochem. 69:95–114, 2000.
Dent, E. W., and F. B. Gertler. Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40(2):209–227, 2003.
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.
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.
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.
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.
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.
Glotzer, M. The 3Ms of central spindle assembly: microtubules, motors and MAPs. Nat. Rev. Mol. Cell Biol. 10(1):9–20, 2009.
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.
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.
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.
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.
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.
Kuchnir Fygenson, D., H. Flyvbjerg, K. Sneppen, A. Libchaber, and S. Leibler. Spontaneous nucleation of microtubules. Phys. Rev. E 51(5):5058–5063, 1995.
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.
Minton, A. P. Excluded volume as a determinant of macromolecular structure and reactivity. Biopolymers 20(10):2093–2120, 1981.
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.
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.
Norholm, M. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10(1):21, 2010.
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.
Odde, D. J. Estimation of the diffusion-limited rate of microtubule assembly. Biophys. J. 73(1):88–96, 1997.
Oosawa, F., and S. Asakura. Thermodynamics of the Polymerization of Protein, Vol. 20. London: Academic Press, 1975.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Wang, H. W., and E. Nogales. Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly. Nature 435(7044):911–915, 2005.
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.
Winey, M., and K. Bloom. Mitotic spindle form and function. Genetics 190(4):1197–1224, 2012.
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.
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.
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.
Michal Wieczorek and Sami Chaaban contributed equally to this work.
Associate Editor William O. Hancock oversaw the review of this article.
About this article
Cite this article
Wieczorek, M., Chaaban, S. & Brouhard, G.J. Macromolecular Crowding Pushes Catalyzed Microtubule Growth to Near the Theoretical Limit. Cel. Mol. Bioeng. 6, 383–392 (2013) doi:10.1007/s12195-013-0292-9
- Excluded volume
- Macromolecular crowding