The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells.
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.
Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch, France.
Anne-Sophie
AS
Rinaldi
Guillaume
G
Freund
Dominique
D
Desplancq
Annie-Paule
AP
Sibler
Mireille
M
Baltzinger
Natacha
N
Rochel
Yves
Y
Mély
Pascal
P
Didier
Etienne
E
Weiss
D021581:Active Transport, Cell Nucleus; D014408:Biomarkers, Tumor / Q000032:analysis / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry*; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes / Q000737:chemistry; D009369:Neoplasms / Q000737:chemistry / Q000175:diagnosis; D010957:Plasmids / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D057127:Single-Chain Antibodies / Q000737:chemistry*; D014162:Transfection
D014408:Biomarkers, Tumor; D005456:Fluorescent Dyes; D007135:Immunoglobulin Variable Region; D046912:Multiprotein Complexes; C403591:PSMD10 protein, human; D011518:Proto-Oncogene Proteins; D057127:Single-Chain Antibodies; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex