Perturbations in Microtubule Mechanics from Tubulin Preparation

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.

[1]  David A Weitz,et al.  Bending dynamics of fluctuating biopolymers probed by automated high-resolution filament tracking. , 2007, Biophysical journal.

[2]  A. Hyman,et al.  Preparation of modified tubulins. , 1991, Methods in enzymology.

[3]  W. Stahel,et al.  Log-normal Distributions across the Sciences: Keys and Clues , 2001 .

[4]  William O. Hancock,et al.  A Polarized Microtubule Array for Kinesin-Powered Nanoscale Assembly and Force Generation , 2002 .

[5]  C. Cantor,et al.  Microtubule assembly in the absence of added nucleotides. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A. Matus,et al.  Domains of Neuronal Microtubule-associated Proteins and Flexural Rigidity of Microtubules , 1997, The Journal of cell biology.

[7]  Matthew Mirigian,et al.  Mechanics of microtubules. , 2010, Journal of biomechanics.

[8]  Linda Z. Shi,et al.  Laser nanosurgery of single microtubules reveals location-dependent depolymerization rates. , 2007, Journal of biomedical optics.

[9]  Youli Li,et al.  Microtubule protofilament number is modulated in a stepwise fashion by the charge density of an enveloping layer. , 2007, Biophysical journal.

[10]  Shannon F. Stewman,et al.  Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration , 2011, Nature Cell Biology.

[11]  H. Erickson,et al.  XMAP215 is a long thin molecule that does not increase microtubule stiffness. , 2001, Journal of cell science.

[12]  Viola Vogel,et al.  Motor-protein "roundabouts": microtubules moving on kinesin-coated tracks through engineered networks. , 2004, Lab on a chip.

[13]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[14]  D. Chrétien,et al.  New data on the microtubule surface lattice , 1991, Biology of the cell.

[15]  B. Mickey,et al.  Rigidity of microtubules is increased by stabilizing agents , 1995, The Journal of cell biology.

[16]  J. Howard,et al.  Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape , 1993, The Journal of cell biology.

[17]  P. Atzberger,et al.  Spectral analysis methods for the robust measurement of the flexural rigidity of biopolymers. , 2012, Biophysical journal.

[18]  Viola Vogel,et al.  Harnessing biological motors to engineer systems for nanoscale transport and assembly. , 2008, Nature nanotechnology.

[19]  Takahiro Nitta,et al.  Dispersion in active transport by kinesin-powered molecular shuttles. , 2005, Nano letters.

[20]  F Metoz,et al.  Lattice defects in microtubules: protofilament numbers vary within individual microtubules , 1992, The Journal of cell biology.

[21]  R. Wade,et al.  How does taxol stabilize microtubules? , 1995, Current Biology.

[22]  M. Magnasco,et al.  Measurement of the persistence length of polymerized actin using fluorescence microscopy. , 1993, Physical review. E, Statistical physics, plasmas, fluids, and related interdisciplinary topics.

[23]  M. Kirschner,et al.  Microtubule bending and breaking in living fibroblast cells. , 1999, Journal of cell science.

[24]  R. Williams,et al.  Taxol-induced flexibility of microtubules and its reversal by MAP-2 and Tau. , 1993, The Journal of biological chemistry.

[25]  G. Borisy,et al.  Conjugation of fluorophores to tubulin , 2005, Nature Methods.

[26]  Erwin Frey,et al.  Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length , 2005, Proceedings of the National Academy of Sciences.

[27]  Shin'ichi Ishiwata,et al.  Temperature dependence of the flexural rigidity of single microtubules. , 2008, Biochemical and biophysical research communications.

[28]  Mary Elizabeth Williams,et al.  Directing transport of CoFe2O4-functionalized microtubules with magnetic fields. , 2007, Small.

[29]  Francesco Pampaloni,et al.  Microtubule Architecture: Inspiration for Novel Carbon Nanotube-based Biomimetic Materials , 2022 .

[30]  T. Mitchison,et al.  Microtubule polymerization dynamics. , 1997, Annual review of cell and developmental biology.

[31]  Erwin Frey,et al.  Microtubule dynamics depart from the wormlike chain model. , 2008, Physical review letters.

[32]  Marileen Dogterom,et al.  A bending mode analysis for growing microtubules: evidence for a velocity-dependent rigidity. , 2004, Biophysical journal.

[33]  R. Tibshirani,et al.  An Introduction to the Bootstrap , 1995 .