Abstract
Microtubules are filamentous biopolymers involved in essential biological processes. They form key structures in eukaryotic cells, and thus it is very important to determine the mechanisms involved in the formation and maintenance of the microtubule network. Microtubule bucklings are transient and localized events commonly observed in living cells and characterized by a fast bending and its posterior relaxation. Active forces provided by molecular motors have been indicated as responsible for most of these rapid deformations. However, the factors that control the shape amplitude and the time scales of the rising and release stages remain unexplored. In this work, we study microtubule buckling in living cells using Xenopus laevis melanophores as a model system. We tracked single fluorescent microtubules from high temporal resolution (0.3–2 s) confocal movies. We recovered the center coordinates of the filaments with 10-nm precision and analyzed the amplitude of the deformation as a function of time. Using numerical simulations, we explored different force mechanisms resulting in microtubule bending. The simulated events reproduce many features observed for microtubules, suggesting that a mechanistic model captures the essential processes underlying microtubule buckling. Also, we studied the interplay between actively transported vesicles and the microtubule network using a two-color technique. Our results suggest that microtubules may affect transport indirectly besides serving as tracks of motor-driven organelles. For example, they could obstruct organelles at microtubule intersections or push them during filament mechanical relaxation.
Similar content being viewed by others
References
Akhmanova A, Steinmetz MO (2015) Control of microtubule organization and dynamics: two ends in the limelight. Nat Rev Mol Cell Biol 16(12):711–726
Bicek AD, Tüzel E, Kroll DM, Odde DJ (2007) Analysis of microtubule curvature. Methods Cell Biol 83:237–268
Bicek AD, Tuzel E, Demtchouk A, Uppalapati M, Hancock WO, Kroll DM, Odde DJ (2009) Anterograde microtubule transport drives microtubule bending in LLC-PK1 epithelial cells. Mol Biol Cell 20(12):2943–2953
Brangwynne CP, MacKintosh FC, Kumar S, Geisse NA, Talbot J, Mahadevan L, Parker KK, Ingber DE, Weitz DA (2006) Microtubules can bear enhanced compressive loads in living cells because of lateral reinforcement. J Cell Biol 173(5):733–741
Brangwynne CP, MacKintosh FC, Weitz DA (2007) Force fluctuations and polymerization dynamics of intracellular microtubules. Proc Natl Acad Sci USA 104(41):16128–16133
Brangwynne CP, Koenderink GH, Mackintosh FC, Weitz DA (2008) Nonequilibrium microtubule fluctuations in a model cytoskeleton. Phys Rev Lett 100(11):118104
Charlebois BD, Schek HT 3rd, Hunt AJ (2010) Nanometer-resolution microtubule polymerization assays using optical tweezers and microfabricated barriers. Methods Cell Biol 95:207–219
Chernick MR (2007) Bootstrap methods: a guide for practitioners and researchers, 2nd edn
Dogterom M, Yurke B (1997) Measurement of the force–velocity relation for growing microtubules. Science 278(5339):856–860
Felgner H, Frank R, Schliwa M (1996) Flexural rigidity of microtubules measured with the use of optical tweezers. J Cell Sci 109(Pt 2):509–516
Fletcher DA, Mullins RD (2010) Cell mechanics and the cytoskeleton. Nature 463(7280):485–492
Gardel ML, Kasza KE, Brangwynne CP, Liu J, Weitz DA (2008) Mechanical response of cytoskeletal networks. Methods Cell Biol 89:487–519
Gauger E, Stark H (2006) Numerical study of a microscopic artificial swimmer. Phys Rev E Stat Nonlinear Soft Matter Phys 74(2 Pt 1):021907
Gittes F, Mickey B, Nettleton J, Howard J (1993) Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape. J Cell Biol 120(4):923–934
Gittes F, Meyhofer E, Baek S, Howard J (1996) Directional loading of the kinesin motor molecule as it buckles a microtubule. Biophys J 70(1):418–429
Gross SP, Tuma MC, Deacon SW, Serpinskaya AS, Reilein AR, Gelfand VI (2002) Interactions and regulation of molecular motors in Xenopus melanophores. J Cell Biol 156(5):855–865
Hendricks AG, Holzbaur EL, Goldman YE (2012) Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors. Proc Natl Acad Sci USA 109(45):18447–18452
Howard J (2001) Mechanics of motor proteins and the cytoskeleton. Sinauer Associates, Inc, Sunderland
Howard J (2006) Elastic and damping forces generated by confined arrays of dynamic microtubules. Phys Biol 3(1):54–66
Howard J (2009) Mechanical signaling in networks of motor and cytoskeletal proteins. Annu Rev Biophys 38:217–234
Jin MZ, Ru CQ (2013) Localized buckling of a microtubule surrounded by randomly distributed cross linkers. Phys Rev E 88:012701
Kabir AMR, Inoue D, Afrin T, Mayama H, Sada K, Kakugo A (2015) Buckling of microtubules on a 2D elastic medium. Sci Rep 5:17222
Kent IA, Rane PS, Dickinson RB, Ladd AJ, Lele TP (2016) Transient pinning and pulling: a mechanism for bending microtubules. PLoS One 11(3):e0151322
Kimura A, Onami S (2005) Computer simulations and image processing reveal length-dependent pulling force as the primary mechanism for C. elegans male pronuclear migration. Dev Cell 8(5):765–775
Kulic IM, Brown AE, Kim H, Kural C, Blehm B, Selvin PR, Nelson PC, Gelfand VI (2008) The role of microtubule movement in bidirectional organelle transport. Proc Natl Acad Sci USA 105(29):10011–10016
Leidel C, Longoria RA, Gutierrez FM, Schubeita GT (2012) Measuring molecular motor forces in vivo: implications for tug-of-war models of bidirectional transport. Biophys J 103(3):492–500
Levi V, Serpinskaya AS, Gratton E, Gelfand V (2006) Organelle transport along microtubules in Xenopus melanophores: evidence for cooperation between multiple motors. Biophys J 90(1):318–327
Mallik R, Carter BC, Lex SA, King SJ, Gross SP (2004) Cytoplasmic dynein functions as a gear in response to load. Nature 427:649–652
Mickey B, Howard J (1995) Rigidity of microtubules is increased by stabilizing agents. J Cell Biol 130(4):909–917
Newman MEJ (2005) Power laws, Pareto distributions and Zipf’s law. Contemp Phys 46(5):28
Nicastro D, Schwartz C, Pierson J, Gaudette R, Porter ME, McIntosh JR (2006) The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313(5789):944–948
Olesen OF, Kawabata-Fukui H, Yoshizato K, Noro N (2002) Molecular cloning of XTP, a tau-like microtubule-associated protein from Xenopus laevis tadpoles. Gene 283(1–2):299–309
Pallavicini C, Levi V, Wetzler DE, Angiolini JF, Benseñor L, Desposito MA, Bruno L (2014) Lateral motion and bending of microtubules studied with a new single-filament tracking routine in living cells. Biophys J 106(12):2625–2635
Paluch EK, Nelson CM, Biais N, Fabry B, Moeller J, Pruitt BL, Kudryasheva G, Rehfeldt F, Federle W (2015) Mechanotransduction: use the force(s). BMC Biol 13:47. doi:10.1186/s12915-015-0150-4
Portran D, Zoccoler M, Gaillard J, Stoppin-Mellet V, Neumann E, Arnal I, Martiel JL, Vantard M (2013) MAP65/Ase1 promote microtubule flexibility. Mol Biol Cell 24(12):1964–1973
Rauch P, Heine P, Goettgens B, Käs JA (2013) Forces from the rear: deformed microtubules in neuronal growth cones influence retrograde flow and advancement. New J Phys 15:015007
Robert A, Herrmann H, Davidson MW, Gelfand VI (2014) Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases. FASEB J 28(7):2879–2890
Rogers SL, Tint IS, Fanapour PC, Gelfand VI (1997) Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro. Proc Natl Acad Sci USA 94(8):3720–3725
Schnitzer MJ, Visscher K, Block SM (2000) Force production by single kinesin motors. Nat Cell Biol 2(10):718–723
Shekhar N, Neelam S, Wu J, Ladd AJ, Dickinson RB, Lele TP (2013) Fluctuating motor forces bend growing microtubules. Cell Mol Bioeng 6(2):120–129
Soppina V, Rai AK, Ramaiya AJ, Barak P, Mallik R (2009) Tug-of-war between dissimilar teams of microtubule motors regulates transport and fission of endosomes. Proc Natl Acad Sci USA 106(46):19381–19386
Walczak CE, Heald R (2008) Mechanisms of mitotic spindle assembly and function. Int Rev Cytol 265:111–158
Wu J, Misra G, Russell RJ, Ladd AJ, Lele TP, Dickinson RB (2011) Effects of dynein on microtubule mechanics and centrosome positioning. Mol Biol Cell 22(24):4834–4841
Yamada S, Wirtz D, Kuo SC (2000) Mechanics of living cells measured by laser tracking microrheology. Biophys J 78(4):1736–1747
Acknowledgements
We are grateful to E. Cerda for fruitful discussions. We acknowledge support from the Agencia Nacional de Promoción Científica y Tecnológica (PICT 2012-0899) and Universidad de Buenos Aires (UBACyT 20020110100074, 20020120200244), Argentina. We also thank Dr. Vladimir I. Gelfand (Northwestern University, Chicago, IL) for providing the cell line used in this work.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Pallavicini, C., Monastra, A., Bardeci, N.G. et al. Characterization of microtubule buckling in living cells. Eur Biophys J 46, 581–594 (2017). https://doi.org/10.1007/s00249-017-1207-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-017-1207-9