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Atomic Force Microscopy and High-Content Analysis: Two Innovative Technologies for Dissecting the Relationship Between Epithelial–Mesenchymal Transition-Related Morphological and Structural Alterations and Cell Mechanical Properties

  • Stephen T. BuckleyEmail author
  • Anthony M. Davies
  • Carsten Ehrhardt
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 784)

Abstract

Epithelial–mesenchymal transition (EMT) is a complex series of cellular reprogramming events culminating in striking alterations in morphology towards an invasive mesenchymal phenotype. Increasingly, evidence suggests that EMT exerts a pivotal role in pathophysiological situations including fibrosis and cancer. Core to these dynamical changes in cellular polarity and plasticity is discrete modifications in cytoskeletal structure. In particular, newly established actin-stress fibres supplant a preceding system of highly organised cortical actin. Although cumulative studies have contributed to elucidation of the detailed signalling pathways that underpin this elaborate molecular process, there remains a deficiency regarding its precise contribution to cellular biomechanics. The advent of atomic force microscopy (AFM) and high-content analysis (HCA) provides two innovative technologies for dissecting the relationship between EMT-related morphological and structural alterations and cell mechanical properties. AFM permits acquisition of high resolution topographical images and detailed analysis of cellular viscoelasticity while HCA facilitates a comprehensive and perspicacious assessment of morphological changes. In combination, they offer the possibility of novel insights into the dynamic traits of transitioning cells. Herein, a detailed protocol describing AFM and HCA techniques for evaluation of transforming growth factor-β1-induced EMT of alveolar epithelial cells is provided.

Key words

Cytoskeleton Epithelial–mesenchymal transition Atomic force microscopy High-content analysis microscopy 

Notes

Acknowledgements

STB is funded by an IRCSET Government of Ireland Postgraduate Scholarship in Science, Engineering and Technology. This work has been funded in part by a Strategic Research Cluster grant (07/SRC/B1154) under the National Development Plan co-funded by EU Structural Funds and Science Foundation Ireland.

References

  1. 1.
    Lee, J.M., Dedhar, S., Kalluri, R., Thompson, E.W. (2006) The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol. 172, 973–81.PubMedCrossRefGoogle Scholar
  2. 2.
    Kalluri, R., Neilson, E.G. (2003) Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest. 112, 1776–84.PubMedGoogle Scholar
  3. 3.
    Guarino, M., Rubino, B., Ballabio, G. (2007) The role of epithelial-mesenchymal transition in cancer pathology. Pathology. 39, 305–18.PubMedCrossRefGoogle Scholar
  4. 4.
    Radisky, D.C., Kenny, P.A., Bissell, M.J. (2007) Fibrosis and cancer: do myofibroblasts come also from epithelial cells via EMT? J Cell Biochem. 101, 830–9.PubMedCrossRefGoogle Scholar
  5. 5.
    Kalluri, R., Weinberg, R.A. (2009) The basics of epithelial-mesenchymal transition. J Clin Invest. 119, 1420–8.PubMedCrossRefGoogle Scholar
  6. 6.
    Savagner, P. (2001) Leaving the neighborhood: molecular mechanisms involved during epithelial-mesenchymal transition. Bioessays. 23, 912–23.PubMedCrossRefGoogle Scholar
  7. 7.
    Wendt, M.K., Allington, T.M., Schiemann, W.P. (2009) Mechanisms of the epithelial-­mesenchymal transition by TGF-beta. Future Oncol. 5, 114568.PubMedCrossRefGoogle Scholar
  8. 8.
    Thoelking, G., Reiss, B., Wegener, J., Oberleithner, H., Pavenstaedt, H., Riethmuller, C. (2010) Nanotopography follows force in TGF-beta1 stimulated epithelium. Nanotechnology. 21, 265102.PubMedCrossRefGoogle Scholar
  9. 9.
    Shahin, V., Barrera, N.P. (2008) Providing unique insight into cell biology via atomic force microscopy. Int Rev Cytol. 265, 227–52.PubMedCrossRefGoogle Scholar
  10. 10.
    Rausch, O. (2006) High content cellular screening. Curr Opin Chem Biol. 10, 316–20.PubMedCrossRefGoogle Scholar
  11. 11.
    Lieber, M., Smith, B., Szakal, A., Nelson-Rees, W., Todaro, G. (1976) A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer. 17, 62–70.PubMedCrossRefGoogle Scholar
  12. 12.
    Small, J., Rottner, K., Hahne, P., Anderson, K.I. (1999) Visualising the actin cytoskeleton. Microsc Res Tech. 47, 3–17.PubMedCrossRefGoogle Scholar
  13. 13.
    Hutter, J.L., Bechhoefer, J. (1993) Calibration of atomic-force microscope tips. Rev Sci Instrum. 64, 1868–73. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Stephen T. Buckley
    • 1
    Email author
  • Anthony M. Davies
    • 2
  • Carsten Ehrhardt
    • 1
  1. 1.School of Pharmacy & Pharmaceutical SciencesTrinity College DublinDublinIreland
  2. 2.Department of Clinical MedicineTrinity College DublinDublinIreland

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