The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

• Published in: Experimental cell research 2013-04, Vol.319 (6), p.838-849
• Main Authors: Rinaldi, Anne-Sophie, Freund, Guillaume, Desplancq, Dominique, Sibler, Annie-Paule, Baltzinger, Mireille, Rochel, Natacha, Mély, Yves, Didier, Pascal, Weiss, Etienne
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; Active Transport, Cell Nucleus; ANTIBODIES; Biomarkers, Tumor - analysis; Biomarkers, Tumor - chemistry; Cancer; Cell Nucleus - chemistry; Cellular biology; Chromatography, Gel; CYTOPLASM; Cytoplasm - chemistry; FLIM–FRET; FLUORESCENCE; Fluorescence Resonance Energy Transfer; Fluorescent Dyes - chemistry; Gankyrin; GELS; Green Fluorescent Proteins - chemistry; HeLa Cells; Humans; Imaging in living cells; Immunoglobulin Variable Region - chemistry; Intrabodies; Intracellular targeting; Life Sciences; Microscopy; Microscopy, Fluorescence; Multiprotein Complexes - chemistry; NEOPLASMS; Neoplasms - chemistry; Neoplasms - diagnosis; Plasmids - chemistry; Proteasome Endopeptidase Complex - chemistry; Protein Binding; Protein Interaction Mapping; PROTEINS; Proto-Oncogene Proteins - chemistry; Single-Chain Antibodies - chemistry; Single-chain Fv; Transfection; TUMOR CELLS
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/j.yexcr.2013.01.011

Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. [Display omitted] ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins.