Real-time analysis of epithelial-mesenchymal transition using fluorescent single-domain antibodies

Sci Rep. 2015 Aug 21;5:13402. doi: 10.1038/srep13402.

Abstract

Vimentin has become an important biomarker for epithelial-mesenchymal transition (EMT), a highly dynamic cellular process involved in the initiation of metastasis and cancer progression. To date there is no approach available to study endogenous vimentin in a physiological context. Here, we describe the selection and targeted modification of novel single-domain antibodies, so-called nanobodies, to trace vimentin in various cellular assays. Most importantly, we generated vimentin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. Following chromobody fluorescence in a cancer-relevant cellular model, we were able for the first time to monitor and quantify dynamic changes of endogenous vimentin upon siRNA-mediated knockdown, induction with TGF-β and modification with Withaferin A by high-content imaging. This versatile approach allows detailed studies of the spatiotemporal organization of vimentin in living cells. It enables the identification of vimentin-modulating compounds, thereby providing the basis to screen for novel therapeutics affecting EMT.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibody Specificity
  • Antigens / metabolism
  • Cell Line, Tumor
  • Cell Survival
  • Computer Systems*
  • Dogs
  • Epithelial-Mesenchymal Transition* / drug effects
  • Fluorescence
  • Gene Knockdown Techniques
  • Humans
  • Madin Darby Canine Kidney Cells
  • Models, Biological
  • Molecular Sequence Data
  • Protein Structure, Tertiary
  • Single-Domain Antibodies / chemistry
  • Single-Domain Antibodies / metabolism*
  • Transforming Growth Factor beta / pharmacology
  • Vimentin / metabolism
  • Withanolides / pharmacology

Substances

  • Antigens
  • Single-Domain Antibodies
  • Transforming Growth Factor beta
  • Vimentin
  • Withanolides
  • withaferin A