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Macromolecular Crowding Pushes Catalyzed Microtubule Growth to Near the Theoretical Limit

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.

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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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Correspondence to Gary J. Brouhard.

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Michal Wieczorek and Sami Chaaban contributed equally to this work.

Associate Editor William O. Hancock oversaw the review of this article.

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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

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Keywords

  • Microtubule
  • Tubulin
  • Polymerization
  • XMAP215
  • EB1
  • Diffusion
  • Excluded volume
  • Macromolecular crowding
  • Isomerization