Advertisement

Analysis of Microtubule Dynamics Heterogeneity in Cell Culture

  • Anara Serikbaeva
  • Anna Tvorogova
  • Sholpan Kauanova
  • Ivan A. Vorobjev
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1745)

Abstract

Microtubules (MTs) are dynamic components of the cytoskeleton playing an important role in a large number of cell functions. Individual MTs in living cells undergo stochastic switching between alternate states of growth, shortening and attenuated phase, a phenomenon known as tempered dynamic instability. Dynamic instability of MTs is usually analyzed by labeling MTs with +TIPs, namely, EB proteins. Tracking of +TIP trajectories allows analyzing MT growth in cells with a different density of MTs. Numerous labs now use +TIP to track growing MTs in a variety of cell cultures. However, heterogeneity of MT dynamics is usually underestimated, and rather small sampling for the description of dynamic instability parameters is often used. The strategy described in this chapter is the method for repetitive quantitative analysis of MT growth rate within the same cell that allows minimization of the variation in MT dynamics measurement. We show that variability in MT dynamics within a cell when using repeated measurements is significantly less than between different cells in the same chamber. This approach allows better estimation of the heterogeneity of cells’ responses to different treatments. To compare the effects of different MT inhibitors, the protocol using normalized values for MT dynamics and repetitive measurements for each cell is employed. This chapter provides detailed methods for analysis of MT dynamics in tissue cultures. We describe protocols for imaging MT dynamics by fluorescent microscopy, contrast enhancement technique, and MT dynamics analysis using triple color-coded display based on sequential subtraction analysis.

Key words

Microtubule dynamics Fluorescent microscopy Dual color-coded display End-binding protein 

References

  1. 1.
    Desai A, Mitchison TJ (1997) Microtubule polymerization dynamics. Annu Rev Cell Dev Biol 13(1):83–117CrossRefPubMedGoogle Scholar
  2. 2.
    Rodionov VI, Gyoeva FK, Tanaka E et al (1993) Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain. J Cell Biol 123(6):1811–1820CrossRefPubMedGoogle Scholar
  3. 3.
    Alberts B et al (2014) Molecular biology of the cell. Garland Science, New YorkGoogle Scholar
  4. 4.
    Lauffenburger DA, Horwitz AF (1996) Cell migration: a physically integrated molecular process. Cell 84(3):359–369CrossRefPubMedGoogle Scholar
  5. 5.
    Etienne-Manneville S (2013) Microtubules in cell migration. Annu Rev Cell Dev Biol 29:471–499CrossRefPubMedGoogle Scholar
  6. 6.
    Broussard JA, Webb DJ, Kaverina I (2008) Asymmetric focal adhesion disassembly in motile cells. Curr Opin Cell Biol 20:85–90CrossRefPubMedGoogle Scholar
  7. 7.
    Fanara P, Husted KH, Selle K et al (2010) Changes in microtubule turnover accompany synaptic plasticity and memory formation in response to contextual fear conditioning in mice. Neuroscience 168(1):167–178CrossRefPubMedGoogle Scholar
  8. 8.
    Atarod D, Eskandari-Sedighi G, Pazhoohi F et al (2015) Microtubule dynamicity is more important than stability in memory formation: an in vivo study. J Mol Neurosci 56(2):313–319CrossRefPubMedGoogle Scholar
  9. 9.
    Dent EW (2017) Of microtubules and memory: implications for microtubule dynamics in dendrites and spines. Mol Biol Cell 28(1):1–8CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Holy TE, Leibler S (1994) Dynamic instability of microtubules as an efficient way to search in space. Proc Natl Acad Sci U S A 91(12):5682–5685CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Jordan MA, Wilson L (2004) Microtubules as a target for anticancer drugs. Nat Rev Cancer 4(4):253–265CrossRefPubMedGoogle Scholar
  12. 12.
    Stearns ME, Wang M (1992) Taxol blocks processes essential for prostate tumor cell (PC-3 ML) invasion and metastases. Cancer Res 52(13):3776–3781PubMedGoogle Scholar
  13. 13.
    Hotchkiss KA, Ashton AW, Mahmood R et al (2002) Inhibition of endothelial cell function in vitro and angiogenesis in vivo by docetaxel (Taxotere): association with impaired repositioning of the microtubule organizing center. Mol Cancer Ther 1(13):1191–1200PubMedGoogle Scholar
  14. 14.
    Hayot C, Farinelle S, De Decker R et al (2002) In vitro pharmacological characterizations of the anti-angiogenic and anti-tumor cell migration properties mediated by microtubule-affecting drugs, with special emphasis on the organization of the actin cytoskeleton. Int J Oncol 21(2):417–426PubMedGoogle Scholar
  15. 15.
    Pasquier E, Honoré S, Braguer D (2006) Microtubule-targeting agents in angiogenesis: where do we stand? Drug Resist Updat 9(1):74–86CrossRefPubMedGoogle Scholar
  16. 16.
    Gliksman NR, Skibbens RV, Salmon ED (1993) How the transition frequencies of microtubule dynamic instability (nucleation, catastrophe, and rescue) regulate microtubule dynamics in interphase and mitosis: analysis using a Monte Carlo computer simulation. Mol Biol Cell 4(10):1035–1050CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Yvon AM, Wadsworth P (1997) Non-centrosomal microtubule formation and measurement of minus end microtubule dynamics in A498 cells. J Cell Sci 110(19):2391–2401PubMedGoogle Scholar
  18. 18.
    Vorobjev IA, Svitkina TM, Borisy GG (1997) Cytoplasmic assembly of microtubules in cultured cells. J Cell Sci 110(21):2635–2645PubMedGoogle Scholar
  19. 19.
    Song Y, Brady ST (2015) Post-translational modifications of tubulin: pathways to functional diversity of microtubules. Trends Cell Biol 25(3):125–136CrossRefPubMedGoogle Scholar
  20. 20.
    Howard J, Hyman AA (2007) Microtubule polymerases and depolymerases. Curr Opin Cell Biol 19(1):31–35CrossRefPubMedGoogle Scholar
  21. 21.
    Dumontet C, Jordan MA (2010) Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat Rev Drug Discov 9(10):790–803CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Schuyler SC, Pellman D (2001) Microtubule “plus-end-tracking proteins”: the end is just the beginning. Cell 105(4):421–424CrossRefPubMedGoogle Scholar
  23. 23.
    Akhmanova A, Steinmetz MO (2008) Tracking the ends: a dynamic protein network controls the fate of microtubule tips. Nat Rev Mol Cell Biol 9(4):309–322CrossRefPubMedGoogle Scholar
  24. 24.
    Walczak CE (2003) The Kin I kinesins are microtubule end-stimulated ATPases. Mol Cell 11(2):286–288CrossRefPubMedGoogle Scholar
  25. 25.
    Moore A, Wordeman L (2004) The mechanism, function and regulation of depolymerizing kinesins during mitosis. Trends Cell Biol 14(10):537–546CrossRefPubMedGoogle Scholar
  26. 26.
    Grigoriev I, Borisy G, Vorobjev I (2006) Regulation of microtubule dynamics in 3T3 fibroblasts by Rho family GTPases. Cell Motil Cytoskeleton 63(1):29–40CrossRefPubMedGoogle Scholar
  27. 27.
    Dogterom M, Kerssemakers JW, Romet-Lemonne G et al (2005) Force generation by dynamic microtubules. Curr Opin Cell Biol 17(1):67–74CrossRefPubMedGoogle Scholar
  28. 28.
    Kinoshita K, Arnal I, Desai A et al (2001) Reconstitution of physiological microtubule dynamics using purified components. Science 294(5545):1340–1343CrossRefPubMedGoogle Scholar
  29. 29.
    Li W, Moriwaki T, Tani T et al (2012) Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin. J Cell Biol 199(5):849–862CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Komarova YA, Vorobjev IA, Borisy GG (2002) Life cycle of MTs: persistent growth in the cell interior, asymmetric transition frequencies and effects of the cell boundary. J Cell Sci 115(17):3527–3539PubMedGoogle Scholar
  31. 31.
    Azarenko O, Okouneva T, Singletary KW et al (2008) Suppression of microtubule dynamic instability and turnover in MCF7 breast cancer cells by sulforaphane. Carcinogenesis 29(12):2360–2368CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Zilberman Y, Ballestrem C, Carramusa L et al (2009) Regulation of microtubule dynamics by inhibition of the tubulin deacetylase HDAC6. J Cell Sci 122(19):3531–3541CrossRefPubMedGoogle Scholar
  33. 33.
    Applegate KT, Besson S, Matov A et al (2011) plusTipTracker: quantitative image analysis software for the measurement of microtubule dynamics. J Struct Biol 176(2):168–184CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Shelden E, Wadsworth P (1993) Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific. J Cell Biol 120:935–945CrossRefPubMedGoogle Scholar
  35. 35.
    Waterman-Storer CM, Salmon ED (1997) Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling. J Cell Biol 139(2):417–434CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Alieva IB, Berezinskaya T, Borisy GG et al (2015) Centrosome nucleates numerous ephemeral microtubules and only few of them participate in the radial array. Cell Biol Int 39(11):1203–1216CrossRefPubMedGoogle Scholar
  37. 37.
    Perez F, Diamantopoulos GS, Stalder R et al (1999) CLIP-170 highlights growing microtubule ends in vivo. Cell 96:517–527CrossRefPubMedGoogle Scholar
  38. 38.
    Bieling P, Laan L, Schek H et al (2007) Reconstitution of a microtubule plus-end tracking system in vitro. Nature 450(7172):1100–1105CrossRefPubMedGoogle Scholar
  39. 39.
    Tirnauer JS, Grego S, Salmon ED et al (2002) EB1–microtubule interactions in Xenopus egg extracts: role of EB1 in microtubule stabilization and mechanisms of targeting to microtubules. Mol Biol Cell 13(10):3614–3626CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Piehl M, Cassimeris L (2003) Organization and dynamics of growing microtubule plus ends during early mitosis. Mol Biol Cell 14(3):916–925CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Schuster M, Kilaru S, Latz M et al (2015) Fluorescent markers of the microtubule cytoskeleton in Zymoseptoria tritici. Fungal Genet Biol 79:141–149CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Zanic M, Stear JH, Hyman AA (2009) EB1 recognizes the nucleotide state of tubulin in the microtubule lattice. PLoS One 4(10):e7585CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Kapoor S, Panda D (2012) Kinetic stabilization of microtubule dynamics by indanocine perturbs EB1 localization, induces defects in cell polarity and inhibits migration of MDA-MB-231 cells. Biochem Pharmacol 83(11):1495–1506CrossRefPubMedGoogle Scholar
  44. 44.
    Matov A, Applegate K, Kumar P et al (2010) Analysis of microtubule dynamic instability using a plus-end growth marker. Nat Methods 7(9):761–768CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Vorobjev IA, Rodionov VI, Maly IV et al (1999) Contribution of plus and minus end pathways to microtubule turnover. J Cell Sci 112(14):2277–2289PubMedGoogle Scholar
  46. 46.
    Gregoretti IV, Margolin G, Alber MS (2006) Insights into cytoskeletal behavior from computational modeling of dynamic microtubules in a cell-like environment. J Cell Sci 119(22):4781–4788CrossRefPubMedGoogle Scholar
  47. 47.
    Mimori-Kiyosue Y, Grigoriev I, Lansbergen G et al (2005) CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex. J Cell Biol 168(1):141–153CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Toso RJ, Jordan MA, Farrell KW et al (1993) Kinetic stabilization of microtubule dynamic instability in vitro by vinblastine. Biochemistry 32(5):1285–1293CrossRefPubMedGoogle Scholar
  49. 49.
    Dhamodharan R, Wadsworth P (1995) Modulation of microtubule dynamic instability in vivo by brain microtubule associated proteins. J Cell Sci 108:1679–1689PubMedGoogle Scholar
  50. 50.
    Sironi L, Solon J, Conrad C et al (2011) Automatic quantification of microtubule dynamics enables RNAi-screening of new mitotic spindle regulators. Cytoskeleton 68(5):266–278CrossRefPubMedGoogle Scholar
  51. 51.
    Garrison AK, Shanmugam M, Leung HC et al (2012) Visualization and analysis of microtubule dynamics using dual color-coded display of plus-end labels. PLoS One 7(11):e50421CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Jordan MA, Kamath K (2007) How do microtubule-targeted drugs work? An overview. Curr Cancer Drug Targets 7(8):730–742CrossRefPubMedGoogle Scholar
  53. 53.
    Castle BT, McCubbin S, Prahl LS et al (2017) Mechanisms of kinetic stabilization by the drugs paclitaxel and vinblastine. Mol Biol Cell 28(9):1238–1257CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Wilson L, Jordan MA (1995) Microtubule dynamics: taking aim at a moving target. Chem Biol 2(9):569–573CrossRefPubMedGoogle Scholar
  55. 55.
    Dorléans A, Gigant B, Ravelli RB et al (2009) Variations in the colchicine-binding domain provide insight into the structural switch of tubulin. Proc Natl Acad Sci U S A 106(33):13775–13779CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Gigant B, Wang C, Ravelli RB et al (2005) Structural basis for the regulation of tubulin by vinblastine. Nature 435(7041):519–522CrossRefPubMedGoogle Scholar
  57. 57.
    Nogales E, Whittaker M, Milligan RA (1999) High-resolution model of the microtubule. Cell 96(1):79–88CrossRefPubMedGoogle Scholar
  58. 58.
    Honore S, Kamath K, Braguer D et al (2004) Synergistic suppression of microtubule dynamics by discodermolide and paclitaxel in non-small cell lung carcinoma cells. Cancer Res 64(14):4957–4964CrossRefPubMedGoogle Scholar
  59. 59.
    Smith JA, Wilson L, Azarenko O et al (2010) Eribulin binds at microtubule ends to a single site on tubulin to suppress dynamic instability. Biochemistry 49(6):1331–1337CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Honore S, Braguer D (2011) Investigating microtubule dynamic instability using microtubule-targeting agents. In: Straube A (ed) Microtubule dynamics: methods and protocols, Methods in molecular biology, vol 777. Springer, New York, pp 245–260CrossRefGoogle Scholar
  61. 61.
    Yang H, Ganguly A, Cabral F (2010) Inhibition of cell migration and cell division correlates with distinct effects of microtubule inhibiting drugs. J Biol Chem 285(42):32242–32250CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Mohan R, Katrukha EA, Doodhi H et al (2013) End-binding proteins sensitize microtubules to the action of microtubule-targeting agents. Proc Natl Acad Sci U S A 110(22):8900–8905CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Yvon AM, Wadsworth P, Jordan MA (1999) Taxol suppresses dynamics of individual microtubules in living human tumor cells. Mol Biol Cell 10(4):947–959CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Pasquier E, Honore S, Pourroy B et al (2005) Antiangiogenic concentrations of paclitaxel induce an increase in microtubule dynamics in endothelial cells but not in cancer cells. Cancer Res 65(6):2433–2440CrossRefPubMedGoogle Scholar
  65. 65.
    Gan PP, McCarroll JA, Po'uha ST (2010) Microtubule dynamics, mitotic arrest, and apoptosis: drug-induced differential effects of βIII-tubulin. Mol Cancer Ther 9(5):1339–1348CrossRefPubMedGoogle Scholar
  66. 66.
    Mikhailov A, Gundersen GG (1998) Relationship between microtubule dynamics and lamellipodium formation revealed by direct imaging of microtubules in cells treated with nocodazole or taxol. Cell Motil Cytoskeleton 41(4):325–340CrossRefPubMedGoogle Scholar
  67. 67.
    Vasquez RJ, Howell B, Yvon AM (1997) Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro. Mol Biol Cell 8(6):973–985CrossRefPubMedPubMedCentralGoogle Scholar
  68. 68.
    Wieczorek M, Bechstedt S, Chaaban S (2015) Microtubule-associated proteins control the kinetics of microtubule nucleation. Nat Cell Biol 17(7):907–916CrossRefPubMedGoogle Scholar
  69. 69.
    Ganguly A, Yang H, Zhang H (2013) Microtubule dynamics control tail retraction in migrating vascular endothelial cells. Mol Cancer Ther 12(12):2837–2846CrossRefPubMedPubMedCentralGoogle Scholar
  70. 70.
    Pourroy B, Honoré S, Pasquier E (2006) Antiangiogenic concentrations of vinflunine increase the interphase microtubule dynamics and decrease the motility of endothelial cells. Cancer Res 66(6):3256–3263CrossRefPubMedGoogle Scholar
  71. 71.
    Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5):2775–2783CrossRefPubMedPubMedCentralGoogle Scholar
  72. 72.
    Wadsworth P (1999) Regional regulation of microtubule dynamics in polarized, motile cells. Cell Motil Cytoskeleton 42(1):48–59CrossRefPubMedGoogle Scholar
  73. 73.
    Heppner GH (1989) Tumor cell societies. J Natl Cancer Inst 81(9):648–649CrossRefPubMedGoogle Scholar
  74. 74.
    Fisher R, Pusztai L, Swanton C (2013) Cancer heterogeneity: implications for targeted therapeutics. Br J Cancer 108(3):479–485CrossRefPubMedPubMedCentralGoogle Scholar
  75. 75.
    Meacham CE, Morrison SJ (2013) Tumour heterogeneity and cancer cell plasticity. Nature 501(7467):328–337CrossRefPubMedPubMedCentralGoogle Scholar
  76. 76.
    Gascoigne KE, Taylor SS (2008) Cancer cells display profound intra-and interline variation following prolonged exposure to antimitotic drugs. Cancer Cell 14(2):111–122CrossRefPubMedGoogle Scholar
  77. 77.
    Joshi HC, Yen TJ, Cleveland DW (1987) In vivo co-assembly of a divergent beta-tubulin subunit (c beta 6) into microtubules of different function. J Cell Biol 105(5):2179–2190CrossRefPubMedGoogle Scholar
  78. 78.
    Lewis SA, Gu W, Cowan NJ (1987) Free intermingling of mammalian β-tubulin isotypes among functionally distinct microtubules. Cell 49(4):539–548CrossRefPubMedGoogle Scholar
  79. 79.
    Lopata MA, Cleveland DW (1987) In vivo microtubules are copolymers of available 13-tubulin isotypes: localization of each of six vertebrate 13-tubulin isotypes using polyclonal antibodies elicited by synthetic peptide antigens. J Cell Biol 105:1707–1720CrossRefPubMedGoogle Scholar
  80. 80.
    Stengel C, Newman SP, Leese MP et al (2010) Class III β-tubulin expression and in vitro resistance to microtubule targeting agents. Br J Cancer 102(2):316–324CrossRefPubMedGoogle Scholar
  81. 81.
    Ganguly A, Yang H, Cabral F (2011) Class III β-tubulin counteracts the ability of paclitaxel to inhibit cell migration. Oncotarget 2(5):368–377CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Anara Serikbaeva
    • 1
    • 5
  • Anna Tvorogova
    • 2
  • Sholpan Kauanova
    • 3
    • 4
  • Ivan A. Vorobjev
    • 5
  1. 1.Department of Biology, School of Science and TechnologyNazarbayev UniversityAstanaKazakhstan
  2. 2.A.N. Belozersky Institute of Physico-Chemical BiologyM.V. Lomonosov Moscow State UniversityMoscowRussia
  3. 3.School of EngineeringNazarbayev UniversityAstanaKazakhstan
  4. 4.National Laboratory AstanaNazarbayev UniversityAstanaKazakhstan
  5. 5.Department of Biology, School of Sciences and TechnologyNazarbayev UniversityAstanaKazakhstan

Personalised recommendations