Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay
Institut de Biologie du Développement de Marseille Luminy, IBDML, UMR 6216, CNRS, Université de la méditerranée, Parc Scientifique de Luminy, Case 907, 13288, Marseille Cedex 09, France
BMC Biology 2011, 9:5 doi:10.1186/1741-7007-9-5Published: 28 January 2011
Additional File 1:
Establishing physiological levels of protein expression with the ultrabithorax (Ubx)-Gal4 driver. (A) The Ubx-Gal4 driver was used to express the green fluorescent protein (GFP) reporter protein (red), showing an expression profile similar to endogenous Ubx protein (grey) in a stage 10 embryo. (B) Establishing physiological levels of VC-Ubx (VCU) expression with the armadillo (arm)-Gal4 driver. The average level of VCU was quantified in the T2 thoracic segment and compared to the level of endogenous Ubx in the A1 segment of a wild type embryo (red-dotted circles). Fluorescent immunostainings were similarly performed with an anti-Ubx antibody (grey). Graph on the right is a boxplot representation of the statistical quantification of the surface and intensity of the fluorescent Ubx immunostaining. It shows that VCU is expressed at around 80% of endogenous Ubx under these conditions. (C) Establishing physiological levels of expression with the Ubx-Gal4 driver. Quantifications were measured with an anti-GFP that recognizes the VC fragment of VCU. Fluorescent immunostainings (grey) were performed in embryos expressing VCU either with arm-Gal4 or Ubx-Gal4 at 29°C. Graph on the right indicates that Ubx-Gal4 led to a slightly better expression than armGal4 (around 20% more). From (B) and (C), we concluded that using Ubx-Gal4 at 29°C allows expression levels comparable to endogenous Ubx levels found in the A1 segment of a wild type embryo.
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Additional File 2. Live imaging of a developing embryo expressing the VC-abdominalA (AbdA) and VN-extradenticle (Exd) fusion proteins with the abdA-Gal4 driver. Live imaging was acquired from stage 10 to stage 14 of embryogenesis.
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Additional File 3:
Influence of fusion topologies on bimolecular fluorescence complementation (BiFC) resulting from extradenticle (Exd)/homothorox (Hth) complex assembly. (A) Schematic representation of Exd and Hth fusion proteins. Interacting domains (PBCA in Exd, HM in Hth) are indicated. (B) BiFC with the indicated fusion proteins which were expressed with the engrailed (en)-Gal4 driver. No signal can be visualized between Hth-VN and VC-Exd. (C) The VC-Hth and Hth-VN fusion proteins are expressed at similar levels with the en-Gal4 driver. Fusion proteins expression was revealed with a polyclonal anti-green fluorescent protein antibody (grey) that recognizes both fragments of Venus. Images were acquired with identical confocal parameters.
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Additional File 4:
Self-assembly properties of the VN and VC fragments in the Drosophila embryo. (A) The VN and VC fragments were expressed with the abdA-Gal4 driver, either as isolated peptides, or in the context of an abdominalA fusion protein, as indicated above pictures. Bimolecular fluorescence complementation (BiFC) was visualized in stage 11 or stage 14 embryos, after 28 h of incubation at 4°C. BiFC resulting from the assembly of isolated Venus (VN) and VC fragments was already visible after a short incubation time of 2 h, but the intensity of the fluorescence did not increase with longer times of incubation (see also the green-dotted curve in Figure 4b). (B) Expression level of the VN and VC fragments, as revealed with a polyclonal anti-green fluorescent protein antibody (grey) that recognizes both fragments. Images were acquired with identical confocal parameters. Note that the VN fragment is more specifically addressed to the nucleus than the VC fragment, due to the addition of a nuclear localization signal (see Methods).
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Additional File 5:
The mutation in the homeodomain (HD) of abdominalA (AbdA) and extradenticle (Exd) does not affect their expression profile and abolishes bimolecular fluorescence complementation (BiFC). (A) The wild-type (VCA) and homeodomain (HD)-mutated (VCAHD) forms of the VC-AbdA fusion protein are expressed at comparable levels in the embryo. Quantifications were performed with an anti-green fluorescent protein antibody (grey) in embryos heterozygous for the PabdAGal4 driver. (B) The wild-type (VNE) and HD-mutated forms (VNEHD) of the HA-tagged VN-Exd fusion proteins are expressed at comparable levels in the embryo. Quantifications were performed with an anti-HA antibody (grey) in embryos heterozygous for the PabdAGal4 driver. Graphs on the right illustrate the statistical quantification as boxplot. (C) The VCAHD and VNEHD fusion proteins did not produced BiFC in vivo. Fusion proteins were expressed with the engrailed (en)-Gal4 driver at 18°C or 29°C. High levels of fusion proteins expression were confirmed by the AbdA (magenta) and Exd (with anti-HA, grey) immunostainings. Despite these high levels of protein expression, no BiFC can be visualized (upper images), highlighting the specificity of the methodology.
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