title: Real-time analysis of epithelial-mesenchymal transition using fluorescent single-domain antibodies.
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.
authors: Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.
authors: Julia
authors: J
authors: Maier
authors: Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.
authors: Bjoern
authors: B
authors: Traenkle
authors: Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.|Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstr. 55, 72770 Reutlingen, Germany.
authors: Ulrich
authors: U
authors: Rothbauer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D000941:Antigens / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival; D003199:Computer Systems; D004285:Dogs; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D005453:Fluorescence; D055785:Gene Knockdown Techniques; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008954:Models, Biological; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014746:Vimentin / Q000378:metabolism; D054358:Withanolides / Q000494:pharmacology
chemical_list: D000941:Antigens; D061905:Single-Domain Antibodies; D016212:Transforming Growth Factor beta; D014746:Vimentin; D054358:Withanolides; C009684:withaferin A