Lateral motion and bending of microtubules studied with a new single-filament tracking routine in living cells.

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells.

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

[2]  W. Webb,et al.  Precise nanometer localization analysis for individual fluorescent probes. , 2002, Biophysical journal.

[3]  J. Fredberg,et al.  Cytoskeleton dynamics: fluctuations within the network. , 2007, Biochemical and biophysical research communications.

[4]  Andrew D. Bicek,et al.  Anterograde microtubule transport drives microtubule bending in LLC-PK1 epithelial cells. , 2009, Molecular biology of the cell.

[5]  Paul R. Selvin,et al.  The role of microtubule movement in bidirectional organelle transport , 2008, Proceedings of the National Academy of Sciences.

[6]  David Zwicker,et al.  Tracking single particles and elongated filaments with nanometer precision. , 2011, Biophysical journal.

[7]  Paul R. Selvin,et al.  Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization , 2003, Science.

[8]  C. Brangwynne,et al.  Force fluctuations and polymerization dynamics of intracellular microtubules , 2007, Proceedings of the National Academy of Sciences.

[9]  Donald E. Ingber,et al.  Jcb: Article Introduction , 2002 .

[10]  R. Goldman,et al.  The dynamic properties of intermediate filaments during organelle transport , 2009, Journal of Cell Science.

[11]  N. Rusan,et al.  Centrosome fragments and microtubules are transported asymmetrically away from division plane in anaphase , 2005, The Journal of cell biology.

[12]  Philip D. Wasserman,et al.  Advanced methods in neural computing , 1993, VNR computer library.

[13]  A. Ladd,et al.  Effects of dynein on microtubule mechanics and centrosome positioning , 2011, Molecular biology of the cell.

[14]  J. Howard,et al.  Mechanics of Motor Proteins and the Cytoskeleton , 2001 .

[15]  Daniel A. Fletcher,et al.  Cell mechanics and the cytoskeleton , 2010, Nature.

[16]  David A Weitz,et al.  Intracellular transport by active diffusion. , 2009, Trends in cell biology.

[17]  Enrico Gratton,et al.  Exploring dynamics in living cells by tracking single particles , 2007, Cell Biochemistry and Biophysics.

[18]  Andrew D. Bicek,et al.  Analysis of microtubule curvature. , 2007, Methods in cell biology.

[19]  T. Svitkina,et al.  Functional coordination of microtubule-based and actin-based motility in melanophores , 1998, Current Biology.

[20]  R Ezzell,et al.  F-actin, a model polymer for semiflexible chains in dilute, semidilute, and liquid crystalline solutions. , 1996, Biophysical journal.

[21]  N. Noro,et al.  Molecular cloning of XTP, a tau-like microtubule-associated protein from Xenopus laevis tadpoles. , 2002, Gene.

[22]  K. Jacobson,et al.  Single-particle tracking: applications to membrane dynamics. , 1997, Annual review of biophysics and biomolecular structure.

[23]  I. Tolic-Nørrelykke,et al.  Anomalous diffusion in living yeast cells. , 2004, Physical review letters.

[24]  M. Kirschner,et al.  Dynamic instability of microtubule growth , 1984, Nature.

[25]  Simon Haykin,et al.  Neural Networks: A Comprehensive Foundation , 1998 .

[26]  M. Schliwa,et al.  Flexural rigidity of microtubules measured with the use of optical tweezers. , 1996, Journal of cell science.

[27]  J. C. Ambrose,et al.  Spatial organization of plant cortical microtubules: close encounters of the 2D kind. , 2009, Trends in cell biology.

[28]  M K Cheezum,et al.  Quantitative comparison of algorithms for tracking single fluorescent particles. , 2001, Biophysical journal.

[29]  Scott D. Hansen,et al.  Differential remodeling of actin cytoskeleton architecture by profilin isoforms leads to distinct effects on cell migration and invasion. , 2012, Cancer cell.

[30]  C. Cohan,et al.  Focal loss of actin bundles causes microtubule redistribution and growth cone turning , 2002, The Journal of cell biology.

[31]  Sarika Sharma,et al.  Desmin and vimentin intermediate filament networks: their viscoelastic properties investigated by mechanical rheometry. , 2009, Journal of molecular biology.

[32]  Vladimir Gelfand,et al.  Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[33]  S. Deacon,et al.  Interactions and regulation of molecular motors in Xenopus melanophores , 2002, The Journal of cell biology.

[34]  Ben Fabry,et al.  Stress fluctuations and motion of cytoskeletal-bound markers. , 2007, Physical review. E, Statistical, nonlinear, and soft matter physics.

[35]  V. Levi,et al.  When size does matter: organelle size influences the properties of transport mediated by molecular motors. , 2013, Biochimica et biophysica acta.

[36]  David A Weitz,et al.  Chapter 19: Mechanical response of cytoskeletal networks. , 2008, Methods in cell biology.

[37]  D. Weihs,et al.  Origin of active transport in breast-cancer cells , 2013 .

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

[39]  V. Levi,et al.  Transition to superdiffusive behavior in intracellular actin-based transport mediated by molecular motors. , 2009, Physical review. E, Statistical, nonlinear, and soft matter physics.

[40]  V. Levi,et al.  Anomalous dynamics of melanosomes driven by myosin-V in Xenopus laevis melanophores. , 2009, Biophysical journal.

[41]  Enrico Gratton,et al.  Organelle transport along microtubules in Xenopus melanophores: evidence for cooperation between multiple motors. , 2006, Biophysical journal.

[42]  J. Lee,et al.  In vitro reconstitution of calf brain microtubules: effects of solution variables. , 1977, Biochemistry.

[43]  E. Mandelkow,et al.  Microtubules and microtubule-associated proteins. , 1995, Current opinion in cell biology.

[44]  M. Koonce,et al.  Pushing forces drive the comet-like motility of microtubule arrays in Dictyostelium. , 2005, Molecular biology of the cell.

[45]  David J. Odde,et al.  Microtubule Tip Tracking and Tip Structures at the Nanometer Scale Using Digital Fluorescence Microscopy , 2011, Cellular and molecular bioengineering.

[46]  J. Bacri,et al.  Magnetic nanomanipulations inside living cells compared with passive tracking of nanoprobes to get consensus for intracellular mechanics. , 2012, Physical review. E, Statistical, nonlinear, and soft matter physics.

[47]  Marcelo Zoccoler,et al.  MAP65/Ase1 promote microtubule flexibility , 2013, Molecular biology of the cell.

[48]  A. Caspi,et al.  Diffusion and directed motion in cellular transport. , 2002, Physical review. E, Statistical, nonlinear, and soft matter physics.

[49]  Vladimir Gelfand,et al.  Myosin cooperates with microtubule motors during organelle transport in melanophores , 1998, Current Biology.

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

[51]  F. MacKintosh,et al.  Nonequilibrium Mechanics of Active Cytoskeletal Networks , 2007, Science.

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

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