Signal transduction systems of cells have been wildly investigated in this decade. Recent studies are focusing attentions on dynamic visualization of signal transduction molecules in living cells. Fluorescence resonance energy transfer (FRET) imaging is powerful microscopic technique to visualize protein-protein interaction in living cells, thus it has been utilized to study signal transduction process. For example, a signal transduction protein fused with two fluorescent proteins (ex. cyan and yellow fluorescent proteins) at C- and N-termini is useful in monitoring the conformational changes of the signal transduction protein in living cells. However, its localization and behavior may vary by fusion of fluorescent proteins or over expression of the fusion protein as compared to those of endogenous signal transduction proteins.

In this study, we attempted to use an enhanced FRET immunoassay method[1], which was developed to analyze antigen concentration with homogeneous assay format. This method can detect intact proteins in vitro using two antibody-fluorescent protein conjugates. In the presence of antigen protein, these conjugates bind to antigen protein and close to each other, and then increase of FRET ratio can be observed. Moreover, to help neighboring of these conjugates, we tethered each conjugate with a leucine zipper motif (c-Jun) at the C-terminus. Here we evaluated whether this method could work in living cells.

F(ab)' fragment of anti-GST antibody and that of antibody recognizing the phosphorylation site of MAPK (ERK1) were conjugated with ECFP-Jun and EYFP-Jun, respectively. Together with GST-ERK1, a model signal transduction molecule, both conjugates were introduced into HeLa cell by microinjection. MAPK signal transduction pathway of HeLa cells was activated by addition of EGF. As a result, GST-ERK1 introduced into the cell was phosphorylated, consequently, increase of FRET ratio from 1.19 to 1.61 was observed in cytoplasm at 6 minutes after addition of EGF. Then FRET ratio decreased but kept an obviously higher value than the base level even after 25 minutes.

By using appropriate fragments of antibodies for target molecules, this method will find a range of applications for real time bio-imaging of endogenous proteins and their behavior specifically in living cells.

Reference: [1]. Y. Ohiro, R. Arai, H. Ueda, T. Nagamune, Analytical Chemistry 74(22): 5786-5792, (2002)