Figure 8.

Multicolour bimolecular fluorescence complementation (BiFC) and simultaneous visualization of multiple protein interactions. (a) BiFC with the red fluorescent protein mCherry. Position of the cut is indicated and was chosen according to previous works in cell cultures [9]. The abdominalA (AbdA) and extradenticle (Exd) fusion proteins were generated with the N-terminal (mCN) or C-terminal (mCC) fragment of mCherry, respectively. BiFC was visualized with the abdA-Gal4 driver. (b) BiFC with the blue fluorescent protein Cerulean. Split Cerulean fragments were generated as in Venus. The AbdA and Exd fusion proteins were constructed with the N-terminal (CN) or C-terminal (CC) fragment of Cerulean, respectively. BiFC was visualized with the abdA-Gal4 driver. (c) Complementation between split fragments of the Venus and Cerulean fluorescent proteins. The VN and CC fragments are able to complement, producing a Venus-like fluorescent signal that is weaker than the one obtained between split fragments of Venus. The CN and VC fragments do not produce BiFC signals, as previously described in cell cultures [7]. Fusion proteins were expressed with the engrailed (en)-Gal4 driver. (d) Multicolour BiFC between ultrabithorax (Ubx), AbdA and Exd proteins fused to split fragments of the Venus and Cerulean proteins, as indicated. All fusion proteins are expressed simultaneously with the en-Gal4 driver. Images of live embryos were acquired separately with specific filters (see Methods). Note that the fluorescence in the middle of the embryo is not specific and corresponds to the auto-fluorescence of the amnioserosa. Auto-fluorescence is particularly strong in the Cerulean spectrum.