Microtubules are essential structures for cellular organization. They support neuronal processes and cilia, they are the scaffolds for the mitotic spindle, and they are the tracks for intracellular transport that actively organizes material and information within the cell. The mechanical properties of microtubules have been studied for almost 30 years, yet the results from different groups are startlingly disparate, ranging over an order of magnitude. Here we present results demonstrating the effects of purification, associated-protein content, age, and fluorescent labeling on the measured persistence length using the freely fluctuating filament method. We find that small percentages (<1%) of residual microtubule-associated proteins left over in the preparation can cause the persistence length to double, and that these proteins also affect the persistence length over time. Interestingly, we find that the fraction of labeled tubulin dimers does not affect the measured persistence length. Further, we have enhanced the analysis method established by previous groups. We have added a bootstrapping with resampling analysis to estimate the error in the variance data used to determine the persistence length. Thus, we are able to perform a weighted fit to the data to more accurately determine the persistence length.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Arnal, I., and R. H. Wade. How does taxol stabilize microtubules? Curr. Biol. 5:900–908, 1995.
Brangwynne, C. P., G. H. Koenderink, E. Barry, Z. Dogic, F. C. MacKintosh, and D. A. Weitz. Bending dynamics of fluctuating biopolymers probed by automated high-resolution filament tracking. Biophys. J. 93:346–359, 2007.
Brown, T. B., and W. O. Hancock. A polarized microtubule array for kinesin-powered nanoscale assembly and force generation. Nano Lett. 2:1131–1135, 2002.
Cassimeris, L., D. Gard, P. T. Tran, and H. P. Erickson. XMAP215 is a long thin molecule that does not increase microtubule stiffness. J. Cell Sci. 114:3025–3033, 2001.
Chrétien, D., F. Metoz, F. Verde, E. Karsenti, and R. H. Wade. Lattice defects in microtubules: protofilament numbers vary within individual microtubules. J. Cell Biol. 117:1031–1040, 1992.
Chrétien, D., and R. H. Wade. New data on the microtubule surface lattice. Biol. Cell 71:161–174, 1991.
Clemmens, J., H. Hess, R. Doot, C. M. Matzke, G. D. Bachand, and V. Vogel. Motor-protein “roundabouts”: microtubules moving on kinesin-coated tracks through engineered networks. Lab Chip 4:83–86, 2004.
Desai, A., and T. J. Mitchison. Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol. 13:83–117, 1997.
Dye, R. B., S. P. Fink, and R. C. Williams. Taxol-induced flexibility of microtubules and its reversal by MAP-2 and Tau. J. Biol. Chem. 268:6847–6850, 1993.
Efron, B., and R. Tibshirani. An Introduction to the Bootstrap. New York: Chapman and Hall, 1993.
Felgner, H., R. Frank, J. Biernat, E. M. Mandelkow, E. Mandelkow, B. Ludin, A. Matus, and M. Schliwa. Domains of neuronal microtubule-associated proteins and flexural rigidity of microtubules. J. Cell Biol. 138:1067–1075, 1997.
Gardner, M. K., B. D. Charlebois, I. M. Jánosi, J. Howard, A. J. Hunt, and D. J. Odde. Rapid microtubule self-assembly kinetics. Cell 146:582–592, 2011.
Gittes, F. Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape. J. Cell Biol. 120:923–934, 1993.
Goel, A., and V. Vogel. Harnessing biological motors to engineer systems for nanoscale transport and assembly. Nat. Nanotechnol. 3:465–475, 2008.
Hawkins, T., M. Mirigian, M. Selcuk Yasar, and J. L. Ross. Mechanics of microtubules. J. Biomech. 43:23–30, 2010.
Hess, S. T., T. P. Girirajan, and M. D. Mason. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91:4258–4272, 2006.
Hutchins, B. M., M. Platt, W. O. Hancock, and M. E. Williams. Directing transport of CoFe2O4-functionalized microtubules with magnetic fields. Small 3:126–131, 2007.
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.
Janson, M. E., and M. Dogterom. A bending mode analysis for growing microtubules: evidence for a velocity-dependent rigidity. Biophys. J. 87:2723–2736, 2004.
Kawaguchi, K., S. Ishiwata, and T. Yamashita. Temperature dependence of the flexural rigidity of single microtubules. Biochem. Biophys. Res. Commun. 366:637–642, 2008.
Kawaguchi, K., and A. Yamaguchi. Temperature dependence rigidity of non-taxol stabilized single microtubules. Biochem. Biophys. Res. Commun. 402:66–69, 2010.
Limpert, E., W. A. Stahel, and M. Abbt. Log-normal distributions across the sciences: keys and clues. Bioscience 51:341–352, 2001.
Mickey, B., and J. Howard. Rigidity of microtubules is increased by stabilizing agents. J. Cell Biol. 130:909–917, 1995.
Nitta, T., and H. Hess. Dispersion in active transport by kinesin-powered molecular shuttles. Nano Lett. 5:1337–1342, 2005.
Odde, D. J., L. Ma, A. H. Briggs, A. DeMarco, and M. W. Kirschner. Microtubule bending and breaking in living fibroblast cells. J. Cell Sci. 112(Pt 19):3283–3288, 1999.
Ott, A., M. Magnasco, A. Simon, and A. Libchaber. Measurement of the persistence length of polymerized actin using fluorescence microscopy. Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics 48:R1642–R1645, 1993.
Pampaloni, F., and E. L. Florin. Microtubule architecture: inspiration for novel carbon nanotube-based biomimetic materials. Trends Biotechnol. 26:302–310, 2008.
Pampaloni, F., G. Lattanzi, A. Jonás, T. Surrey, E. Frey, and E. L. Florin. Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length. Proc. Natl Acad. Sci. USA 103:10248–10253, 2006.
Peloquin, J., Y. Komarova, and G. Borisy. Conjugation of fluorophores to tubulin. Nat. Methods 2:299–303, 2005.
Politis, D. N., and J. P. Romano. Subsampling. New York: Springer, 1999.
R Development Code Team. R Code, 2011.
Raviv, U., T. Nguyen, R. Ghafouri, D. J. Needleman, Y. Li, H. P. Miller, L. Wilson, R. F. Bruinsma, and C. R. Safinya. Microtubule protofilament number is modulated in a stepwise fashion by the charge density of an enveloping layer. Biophys. J. 92:278–287, 2007.
Shelanski, M. L., F. Gaskin, and C. R. Cantor. Microtubule assembly in the absence of added nucleotides. Proc. Natl Acad. Sci. USA 70:765–768, 1973.
Taute, K. M., F. Pampaloni, E. Frey, and E. L. Florin. Microtubule dynamics depart from the wormlike chain model. Phys. Rev. Lett. 100:028102, 2008.
Valdman, D., P. J. Atzberger, D. Yu, S. Kuei, and M. T. Valentine. Spectral analysis methods for the robust measurement of the flexural rigidity of biopolymers. Biophys. J. 102:1144–1153, 2012.
Wakida, N. M., C. S. Lee, E. T. Botvinick, L. Z. Shi, A. Dvornikov, and M. W. Berns. Laser nanosurgery of single microtubules reveals location-dependent depolymerization rates. J. Biomed. Opt. 12:024022, 2007.
Zhang, D., K. D. Grode, S. F. Stewman, J. D. Diaz-Valencia, E. Liebling, U. Rath, T. Riera, J. D. Currie, D. W. Buster, A. B. Asenjo, H. J. Sosa, J. L. Ross, A. Ma, S. L. Rogers, and D. J. Sharp. Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration. Nat. Cell Biol. 13:361–370, 2011.
TLH was supported in part from the North East Alliance for Graduate Education and Professoriate (NEAGEP) grant from the NSF. TLH, MM, and MSY were supported on an NSF grant #1039403 and supplement #0928540 to JLR and DS from the Nano and Bio Mechanics Program, Civil Mechanical, and Manufacturing Innovation Directorate. DLS was supported by funds from the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. We thank Carey Fagerstrom for her preparation of the in-house tubulin and helpful discussions. We thank John Crocker for valuable discussions on statistics and log-normal data sets.
Associate Editor William O. Hancock oversaw the review of this article.
About this article
Cite this article
Hawkins, T.L., Mirigian, M., Li, J. et al. Perturbations in Microtubule Mechanics from Tubulin Preparation. Cel. Mol. Bioeng. 5, 227–238 (2012). https://doi.org/10.1007/s12195-012-0229-8
- Flexural rigidity
- Bending stiffness
- Cytoskeletal network