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Mitosis pp 247-260 | Cite as

Seeded Microtubule Growth for Cryoelectron Microscopy of End-Binding Proteins

  • Sebastian P. Maurer
  • Franck J. Fourniol
  • Andreas Hoenger
  • Thomas SurreyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1136)

Abstract

End-binding proteins (EBs) have the ability to autonomously track the ends of growing microtubules, where they recruit several proteins that control various aspects of microtubule cytoskeleton organization and function. The structural nature of the binding site recognized by EBs at growing microtubule ends has been a subject of debate. Recently, a fluorescence microscopy assay used for the study of dynamic end tracking in vitro was adapted for cryoelectron microscopy (cryo-EM). In combination with single-particle reconstruction methods, this modified assay was used to produce the first subnanometer-resolution model of how the microtubule-binding domain of EBs binds to microtubules grown in the presence of GTPγS. A GTPγS microtubule can be considered a static mimic of the transiently existing binding region of EBs at a microtubule end growing in the presence of GTP. Here we describe in detail the procedure used to generate these samples. It relies on the polymerization of microtubules from preformed stabilized and quantum dot-labeled microtubule seeds. This allows the cryo-EM analysis of proteins bound to paclitaxel-free microtubules. It provides freedom for using different GTP analogues during microtubule elongation independent of their nucleation properties. This assay could also be useful for the cryo-EM analysis of other microtubule-associated proteins.

Key words

cryo-EM Microtubule structure GMP-CPP seeds GTPγS EB1 Mal3 Kinesin 

Notes

Acknowledgements

We thank Julia Cope, Rachel A. Santarella, and Cindi L. Schwartz for training in electron microscope operation and cryoelectron microscope sample preparation. We thank the electron microscopy facility at EMBL Heidelberg for support. S.P.M. was supported by Marie Curie (PIEF-GA-2009-253043) and EMBO (ALTF 1032-2009) fellowships. F.J.F. was supported by an EMBO Long-Term Fellowship (ALTF 219-2011). A.H. and the Boulder 3-D lab are supported by grant P41-GM103431 (NIH-NIGMS).

References

  1. 1.
    Amos LA, Hirose K (2007) Studying the structure of microtubules by electron microscopy. Methods Mol Med 137:65–91PubMedCrossRefGoogle Scholar
  2. 2.
    Fourniol FJ, Moores CA (2011) Snapshots of kinesin motors on microtubule tracks. Methods Mol Biol 778:57–70PubMedCrossRefGoogle Scholar
  3. 3.
    Hoenger A, Gross H (2008) Structural investigations into microtubule-MAP complexes. Methods Cell Biol 84:425–444PubMedCrossRefGoogle Scholar
  4. 4.
    Li H, DeRosier DJ, Nicholson WV, Nogales E, Downing KH (2002) Microtubule structure at 8 A resolution. Structure 10:1317–1328PubMedCrossRefGoogle Scholar
  5. 5.
    Kikkawa M, Hirokawa N (2006) High-resolution cryo-EM maps show the nucleotide binding pocket of KIF1A in open and closed conformations. EMBO J 25:4187–4197PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Hirose K, Akimura E, Akiba T, Endow SA, Amos LA (2006) Large conformational changes in a kinesin motor catalyzed by interaction with microtubules. Mol Cell 23:913–923PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Sindelar CV, Downing KH (2007) The beginning of kinesin’s force-generating cycle visualized at 9-Å resolution. J Cell Biol 177:377–385PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Dietrich KA, Sindelar CV, Brewer PD, Downing KH, Cremo CR, Rice SE (2008) The kinesin-1 motor protein is regulated by a direct interaction of its head and tail. Proc Natl Acad Sci U S A 105:8938–8943PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Bodey AJ, Kikkawa M, Moores CA (2009) 9-Angstrom structure of a microtubule-bound mitotic motor. J Mol Biol 388:218–224PubMedCrossRefGoogle Scholar
  10. 10.
    Peters C, Brejc K, Belmont L, Bodey AJ, Lee Y, Yu M, Guo J, Sakowicz R, Hartman J, Moores CA (2010) Insight into the molecular mechanism of the multitasking kinesin-8 motor. EMBO J 29:3437–3447PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Fourniol FJ, Sindelar CV, Amigues B, Clare DK, Thomas G, Perderiset M, Francis F, Houdusse A, Moores CA (2010) Template-free 13-protofilament microtubule-MAP assembly visualized at 8 A resolution. J Cell Biol 191:463–470PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Alushin GM, Ramey VH, Pasqualato S, Ball DA, Grigorieff N, Musacchio A, Nogales E (2010) The Ndc80 kinetochore complex forms oligomeric arrays along microtubules. Nature 467:805–810PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Sui H, Downing KH (2010) Structural basis of interprotofilament interaction and lateral deformation of microtubules. Structure 18:1022–1031PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Sindelar CV, Downing KH (2010) An atomic-level mechanism for activation of the kinesin molecular motors. Proc Natl Acad Sci U S A 107:4111–4116PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Goulet A, Behnke-Parks WM, Sindelar CV, Major J, Rosenfeld SS, Moores CA (2012) The structural basis of force generation by the mitotic motor kinesin-5. J Biol Chem 287(53):44654–44666PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Maurer SP, Fourniol FJ, Bohner G, Moores CA, Surrey T (2012) EBs recognize a nucleotide-dependent structural cap at growing microtubule ends. Cell 149:371–382PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Redwine WB, Hernández-López R, Zou S, Huang J, Reck-Peterson SL, Leschziner AE (2012) Structural basis for microtubule binding and release by dynein. Science 337:1532–1536PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Yajima H, Ogura T, Nitta R, Okada Y, Sato C, Hirokawa N (2012) Conformational changes in tubulin in GMPCPP and GDP-taxol microtubules observed by cryoelectron microscopy. J Cell Biol 198:315–322PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Bieling P, Telley IA, Hentrich C, Piehler J, Surrey T (2010) Fluorescence microscopy assays on chemically functionalized surfaces for quantitative imaging of microtubule, motor, and +TIP dynamics. Methods Cell Biol 95:555–580PubMedCrossRefGoogle Scholar
  20. 20.
    Gell C, Bormuth V, Brouhard GJ, Cohen DN, Diez S, Friel CT, Helenius J, Nitzsche B, Petzold H, Ribbe J et al (2010) Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy. Methods Cell Biol 95:221–245PubMedCrossRefGoogle Scholar
  21. 21.
    Toomre D, Bewersdorf J (2010) A new wave of cellular imaging. Annu Rev Cell Dev Biol 26:285–314PubMedCrossRefGoogle Scholar
  22. 22.
    Vale RD, Coppin CM, Malik F, Kull FJ, Milligan RA (1994) Tubulin GTP hydrolysis influences the structure, mechanical properties, and kinesin-driven transport of microtubules. J Biol Chem 269:23769–23775PubMedGoogle Scholar
  23. 23.
    Elie-Caille C, Severin F, Helenius J, Howard J, Muller DJ, Hyman AA (2007) Straight GDP-tubulin protofilaments form in the presence of taxol. Curr Biol 17:1765–1770PubMedCrossRefGoogle Scholar
  24. 24.
    Maurer SP, Bieling P, Cope J, Hoenger A, Surrey T (2011) GTPgammaS microtubules mimic the growing microtubule end structure recognized by end-binding proteins (EBs). Proc Natl Acad Sci U S A 108:3988–3993PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Zanic M, Stear JH, Hyman AA, Howard J (2009) EB1 recognizes the nucleotide state of tubulin in the microtubule lattice. PLoS One 4:e7585PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Chretien D, Fuller SD, Karsenti E (1995) Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates. J Cell Biol 129:1311–1328PubMedCrossRefGoogle Scholar
  27. 27.
    Akhmanova A, Steinmetz MO (2010) Microtubule + TIPs at a glance. J Cell Sci 123:3415–3419PubMedCrossRefGoogle Scholar
  28. 28.
    Galjart N (2010) Plus-end-tracking proteins and their interactions at microtubule ends. Curr Biol 20:R528–R537PubMedCrossRefGoogle Scholar
  29. 29.
    Kumar P, Wittmann T (2012) +TIPs: SxIPping along microtubule ends. Trends Cell Biol 22:418–428PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Duellberg C, Fourniol FJ, Maurer SP, Roostalu J, Surrey T (2012) End-binding proteins and Ase1/PRC1 define local functionality of structurally distinct parts of the microtubule cytoskeleton. Trends Cell Biol 23(2):54–63PubMedCrossRefGoogle Scholar
  31. 31.
    Bieling P, Laan L, Schek H, Munteanu EL, Sandblad L, Dogterom M, Brunner D, Surrey T (2007) Reconstitution of a microtubule plus-end tracking system in vitro. Nature 450:1100–1105PubMedCrossRefGoogle Scholar
  32. 32.
    Bieling P, Kandels-Lewis S, Telley IA, van Dijk J, Janke C, Surrey T (2008) CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites. J Cell Biol 183:1223–1233PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Honnappa S, Gouveia SM, Weisbrich A, Damberger FF, Bhavesh NS, Jawhari H, Grigoriev I, van Rijssel FJ, Buey RM, Lawera A et al (2009) An EB1-binding motif acts as a microtubule tip localization signal. Cell 138:366–376PubMedCrossRefGoogle Scholar
  34. 34.
    Castoldi M, Popov AV (2003) Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr Purif 32:83–88PubMedCrossRefGoogle Scholar
  35. 35.
    Hyman A, Drechsel D, Kellogg D, Salser S, Sawin K, Steffen P, Wordeman L, Mitchison T (1991) Preparation of modified tubulins. Methods Enzymol 196:478–485PubMedCrossRefGoogle Scholar
  36. 36.
    Crevel IM, Nyitrai M, Alonso MC, Weiss S, Geeves MA, Cross RA (2004) What kinesin does at roadblocks: the coordination mechanism for molecular walking. EMBO J 23:23–32PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    McIntosh JR, Morphew MK, Grissom PM, Gilbert SP, Hoenger A (2009) Lattice structure of cytoplasmic microtubules in a cultured mammalian cell. J Mol Biol 394:177–182PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Sebastian P. Maurer
    • 1
  • Franck J. Fourniol
    • 1
  • Andreas Hoenger
    • 2
  • Thomas Surrey
    • 1
    Email author
  1. 1.London Research Institute, Cancer Research UKLondonUK
  2. 2.Department of Molecular Cell and Developmental BiologyUniversity of Colorado at BoulderBoulderUSA

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