Skip to main content

Resolving the molecular structure of microtubules under physiological conditions with scanning force microscopy

European Biophysics Journal volume 33, pages 462–467 (2004)Cite this article

Abstract

We have imaged microtubules, essential structural elements of the cytoskeleton in eukaryotic cells, in physiological conditions by scanning force microscopy. We have achieved molecular resolution without the use of cross-linking and chemical fixation methods. With tip forces below 0.3 nN, protofilaments with ~6 nm separation could be clearly distinguished. Lattice defects in the microtubule wall were directly visible, including point defects and protofilament separations. Higher tip forces destroyed the top half of the microtubules, revealing the inner surface of the substrate-attached protofilaments. Monomers could be resolved on these inner surfaces.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price includes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1A, B
Fig. 2A, B
Fig. 3
Fig. 4A–C
Fig. 5A–D

Abbreviations

APTS:

(3-aminopropyl)triethoxysilane

DETA:

N 1-[3-(trimethoxysilyl)propyl]diethylenetriamine

EM:

electron microscopy

MT:

microtubule

SFM:

scanning force microscopy

References

  • Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002) Molecular biology of the cell, 4th edn. Garland, New York

  • Arnal I, Wade RH (1995) How does Taxol stabilize microtubules? Curr Biol 5:900–908

    CAS  PubMed  Google Scholar 

  • Binnig G, Quate CF, Gerber C (1986) Atomic force microscope. Phys Rev Lett 56:930–933

    Article  PubMed  Google Scholar 

  • Cassimeris L, Spittle C (2001) Regulation of microtubule-associated proteins. Int Rev Cytol 210:163–226

    CAS  PubMed  Google Scholar 

  • Chretien D, Metoz F, Verde F, Karsenti E, Wade RH (1992) Lattice defects in microtubules: protofilament numbers vary within individual microtubules. J Cell Biol 117:1031–1040

    CAS  PubMed  Google Scholar 

  • Davis LJ, Odde DJ, Block SM, Gross SP (2002) The importance of lattice defects in katanin-mediated microtubule severing in vitro. Biophys J 82:2916–2927

    CAS  PubMed  Google Scholar 

  • de Pablo PJ, Colchero J, Gomez-Herrero J, Baro AM (1998) Jumping mode scanning force microscopy. Appl Phys Lett 73:3300–3302

    Article  Google Scholar 

  • de Pablo PJ, Schaap IAT, MacKintosh FC, Schmidt CF (2003a) Deformation and collapse of microtubules on the nanometer scale. Phys Rev Lett 91:098101

    PubMed  Google Scholar 

  • de Pablo PJ, Schaap IAT, Schmidt CF (2003b) Observation of microtubules with scanning force microscopy in liquid. Nanotechnology 14:143–146

    Article  Google Scholar 

  • Fritz M, Radmacher M, Allersma MW, Cleveland JP, Stewart RJ, Hansma PK, Schmidt CF (1995) Imaging microtubules in buffer solution using tapping mode atomic force microscopy. Proc SPIE 2384:150–157

    CAS  Google Scholar 

  • Gittes F, Schmidt CF (1998) Thermal noise limitations on micromechanical experiments. Eur Biophys J 27:75–81

    Article  CAS  Google Scholar 

  • Hirokawa N (1998) Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279:519–526

    Article  CAS  PubMed  Google Scholar 

  • Kikkawa M, Ishikawa T, Nakata T, Wakabayashi T, Hirokawa N (1994) Direct visualization of the microtubule lattice seam both in vitro and in vivo. J Cell Biol 127:1965–1971

    CAS  PubMed  Google Scholar 

  • Kirschner MW, Honig LS, Williams RC (1975) Quantitative electron microscopy of microtubule assembly in vitro. J Mol Biol 99:263–276

    CAS  PubMed  Google Scholar 

  • Mandelkow EM, Schultheiss R, Rapp R, Muller M, Mandelkow E (1986) On the surface lattice of microtubules: helix starts, protofilament number, seam, and handedness. J Cell Biol 102:1067–1073

    CAS  PubMed  Google Scholar 

  • McNally FJ, Vale RD (1993) Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell 75:419–429

    CAS  PubMed  Google Scholar 

  • Nogales E, Wolf SG, Downing KH (1998) Structure of the alpha beta tubulin dimer by electron crystallography. Nature 391:199–203

    CAS  PubMed  Google Scholar 

  • Nogales E, Whittaker M, Milligan RA, Downing KH (1999) High-resolution model of the microtubule. Cell 96:79–88

    CAS  PubMed  Google Scholar 

  • Pierson GB, Burton PR, Himes RH (1978) Alterations in number of protofilaments in microtubules assembled in vitro. J Cell Biol 76:223–228

    CAS  PubMed  Google Scholar 

  • Simon JR, Salmon ED (1990) The structure of microtubule ends during the elongation and shortening phases of dynamic instability examined by negative-stain electron microscopy. J Cell Sci 96:571–582

    CAS  PubMed  Google Scholar 

  • Snyder JP, Nettles JH, Cornett B, Downing KH, Nogales E (2001) The binding conformation of Taxol in beta-tubulin: a model based on electron crystallographic density. Proc Natl Acad Sci USA 98:5312–5306

    Article  CAS  PubMed  Google Scholar 

  • Tilney LG, Bryan J, Bush DJ, Fujiwara K, Mooseker MS, Murphy DB, Snyder DH (1973) Microtubules: evidence for 13 protofilaments. J Cell Biol 59:267–275

    CAS  PubMed  Google Scholar 

  • Turner DC, Chang C, Fang K, Brandow SL, Murphy DB (1995) Selective adhesion of functional microtubules to patterned silane surfaces. Biophys J 69:2782–2789

    CAS  PubMed  Google Scholar 

  • Villarrubia JS (1997) Algorithms for scanned probe microscope image simulation, surface reconstruction, and tip estimation. J Res Natl Inst Stand Technol 102:425–454

    Google Scholar 

  • Vinckier A, Dumortier C, Engelborghs Y, Hellemans L (1996) Dynamical and mechanical study of immobilized microtubules with atomic force microscopy. J Vac Sci Technol B 14:1427–1431

    Article  CAS  Google Scholar 

  • Williams RC Jr, Lee JC (1982) Preparation of tubulin from brain. Methods Enzymol 85:376–385

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Ken Downing for sharing his 3D MT model, Per Bullough for help with image analysis, Fred MacKintosh for helpful discussions, Johanna van Nes and René Koops for help with exploratory experiments, and financial support of the Dutch Foundation for Fundamental Research of Matter (FOM) and ALW/FOM project no. 01FB28/2.

Author information

Author notes

  1. Pedro J. de Pablo

    Present address: Departamento de Física de la Materia Condensada C-III, Universidad Autónoma de Madrid, 28049 , Madrid, Spain

Authors and Affiliations

  1. Section Physics of Complex Systems, Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV , Amsterdam, The Netherlands

    Iwan A. T. Schaap, Pedro J. de Pablo & Christoph F. Schmidt

Authors
  1. Iwan A. T. Schaap
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pedro J. de Pablo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christoph F. Schmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Iwan A. T. Schaap.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Schaap, I.A.T., de Pablo, P.J. & Schmidt, C.F. Resolving the molecular structure of microtubules under physiological conditions with scanning force microscopy. Eur Biophys J 33, 462–467 (2004). https://doi.org/10.1007/s00249-003-0386-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00249-003-0386-8

Keywords

  • Atomic force microscopy
  • Microtubule
  • Scanning force microscopy