Generation of a versatile BiFC ORFeome library for analyzing protein–protein interactions in live Drosophila
- University of Zurich, Switzerland
- CNRS, ENS Lyon, France
- Zhejiang University, China
- Aix-Marseille Université, France
- Centre National de la Recherche Scientifique, France
Figures
Figure 1 with 1 supplement
Generation of a Gal4 inducible library compatible with Venus-based BiFC in Drosophila.
(A-A’) Principle of the Venus-based BiFC between a candidate ORF (Open Reading Frame) and a bait protein fused to the N- (VN) or C-terminal (VC) fragment of Venus, respectively. Excitation and …
Figure 1—figure supplement 1
Comparison of Venus-based BiFC when using ORFs swapped with the original VN or VN-short tag line.
(A) Schematic representation of an ORF fused to VN with the original linker region (red arrow). (A’) Illustrative confocal captures of BiFC resulting from the co-expression of ORFs fused to VN with …
Figure 2 with 2 supplements
Generation of a Gal4 inducible library compatible with Venus- and Cerulean-based BiFC in Drosophila.
(A-A’) Principle of bicolor BiFC by using the complementation property between the C-terminal fragment of the blue fluorescent protein Cerulean (CC) and the N-terminal fragment of Venus (VN) or …
Figure 2—figure supplement 1
The 21F and 86Fb attP sites lead to similar expression levels.
. Illustrative confocal capture of UAS-driven GFP inserted in the 21F (A) or 86Fb (B) attP sites with the Ubx-Gal4 driver. Two different stages are shown.
Figure 2—figure supplement 2
Co-expression of non-complementing ORF-3xHA can compete against BiFC signals obtained from ORF-CC and VN-AbdA constructs.
(A) Principle of the BiFC competition experiment with co-expression of a non-complementing HA-tagged ORF. (B) Illustrative confocal captures of BiFC between VN-AbdA and the different ORF-CC …
Figure 3
The TF-3xHA and multicolour BiFC libraries.
(A) Number of transcription factors (TFs) tagged with three repeats of the hemagglutinin (3xHA) epitope at the 3’ end and multicolour BiFC fly strains at FlyORF. Various insertion sites were used …
Figure 4 with 10 supplements
Using the multicolor BiFC library for a large-scale interaction screen with Ubx and AbdA in the live Drosophila embryo.
(A) Number of TFs that were positive (green) or negative (red) with Ubx. (B) Number of TFs that were positive (green) or negative (red) with AbdA. (C) Repartition of the different families among the …
Figure 4—figure supplement 1
Interactomes and negatomes of Ubx and AbdA with the 260 tested TFs.
(A) Schematic representation of Ubx and AbdA. The homeodomain (HD), hexapeptide (HX) and UbdA (UA) motifs are indicated, as well as the percentage of identity of Ubx with AbdA along the …
Figure 4—figure supplement 2
Illustrative confocal captures of Venus-based BiFC between ORF-VN and VC-Ubx, as indicated.
Fusion proteins are expressed with the Ubx-Gal4 driver and BiFC analysed in the epidermis of stage 10 embryos.
Figure 4—figure supplement 3
Illustrative confocal captures of Venus-based BiFC between ORF-CC and VN-Ubx, as indicated.
Fusion proteins are expressed with the Ubx-Gal4 driver and BiFC analysed in the epidermis of stage 10 embryos.
Figure 4—figure supplement 4
Illustrative confocal captures of Venus-based BiFC between ORF-VN and VC-AbdA, as indicated.
Fusion proteins are expressed with the abdA-Gal4 driver and BiFC analysed in the epidermis of stage 10 embryos.
Figure 4—figure supplement 5
Illustrative confocal captures of Venus-based BiFC between ORF-CC and VN-AbdA, as indicated.
Fusion proteins are expressed with the abdA-Gal4 driver and BiFC analysed in the epidermis of stage 10 embryos.
Figure 4—figure supplement 6
Illustrative confocal captures of Cerulean-based BiFC between ORF-CC and CN-AbdA, as indicated.
Fusion proteins are expressed with the abdA-Gal4 driver and BiFC analysed in the epidermis of stage 10 embryos.
Figure 4—figure supplement 7
Representation of the common interactome and negatome between Ubx and AbdA.
Colour code and representation are as in Figure 4—figure supplement 1.
Figure 4—figure supplement 8
Comparison between Interactomes.
(A) Representation of the common and specific interactions among the interactomes of Ubx and AbdA. The corresponding percentage among the overall 184 positive interactions is indicated. (B) …
Figure 4—figure supplement 9
Representation of the Ubx- (A) and AbdA-specific (B) interactomes.
Colour code is as in Figure 3. TFs involved in gonad mesoderm or oenocytes specification, which correspond to AbdA-specific functions, are surrounded by a solid or dotted line, respectively. Star * …
Figure 4—figure supplement 10
Analysis of the co-expression and interaction status of Ubx (A) or AbdA (B) and the positive ORFs.
The distributions of the values taken by the ratio between the number of tissues in which the TF and Ubx or AbdA are co-expressed and the total number of tissues composing the TF expression domain …
Figure 5
Functional genetics validates BiFC observations with Ubx in haltere primordium.
(A) Scanning electron microscopy of haltere phenotypes in the different genetic backgrounds, as indicated. Compared to wild-type, halteres of individuals heterozygous for the Hox regulatory mutation …
Figure 6 with 3 supplements
Using the multicolour BiFC library for analysing two different interactions in the same embryo.
(A) Principle of the bicolour BiFC. The AbdA and Extradenticle (Exd) cofactor are respectively fused to the CN or VN fragment, which can complement with the CC fragment of a co-expressed ORF when …
Figure 6—figure supplement 1
Illustrative confocal pictures of BiFC between ORF-VN and VC-Exd, as indicated.
Fusion proteins are expressed with the abdA-Gal4 driver and BiFC analysed in the epidermis of stage 10 embryos.
Figure 6—figure supplement 2
Interaction properties of Extradenticle (Exd) with a set of 37 TFs that are positive with AbdA.
(A) Representation of the interactome (positive interactions) of Exd. (B) Representation of the negatome (negative interactions) of Exd.
Figure 6—figure supplement 3
Adding the sfGFP to the fluorescence repertoire of the multicolour BiFC library.
(A) Split sfGFP fragments that are normally used for BiFC. (A’) Principle of BiFC with the N-terminal fragment of sfGFP (sfGFPN) and the C-terminal fragment of Cerulean (CC). Excitation and emission …
Figure 7
Coupling the multicolor BiFC library to the split-Gal4 system to visualize interactions in the overlapping expression domain of the two protein partners.
(A) Principle of BiFC with a unique Gal4 driver reproducing the expression profile of the bait protein (for example Hox-Gal4 driver). (B) Principle of BiFC with a Gal4 driver reproducing the …
Additional files
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Supplementary file 1
List of the 235 TFs of the multicolor BiFC library fused to the VN fragment at the C- or N-terminus, with a long or short linker region, as indicated.
Note that several TFs are fused to VN with either a long or a short linker region.
- https://doi.org/10.7554/eLife.38853.025
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Supplementary file 2
List of the 326 TFs of the multicolor BiFC library fused to the CC fragment.
TFs highlighted in light green are also available as VN fusion constructs (see Supplementary file 1).
- https://doi.org/10.7554/eLife.38853.026
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Supplementary file 3
List of the 260 VN- and CC-fusion TFs of the multicolor BiFC library that were tested with Ubx and AbdA.
- https://doi.org/10.7554/eLife.38853.027
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Supplementary file 4
Expression profile of the 260 TFs that were tested with Ubx and AbdA among 25 different developmental contexts of the Drosophila embryo.
Each color code corresponds to a different tissue when expressed. Black boxes depict expression in tissues where Ubx and AbdA are not present. The last two columns indicate the number of co-occurrences of the TF and the Hox protein with regard to the total distribution (parentheses).
- https://doi.org/10.7554/eLife.38853.028
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Supplementary file 5
Analysis of the proportion of common and specific AbdA- and Ubx interactors expressed in the different tissues, as indicated.
- https://doi.org/10.7554/eLife.38853.029
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Supplementary file 6
List of TFs tested in RNAi in Ubx heterozygous mutant haltere discs.
Green and red boxes correspond to increased or not increased RNAi phenotypes, respectively. Black boxes correspond to RNAi phenotypes that could not be interpreted with regard to a potential Ubx cofactor function in the haltere disc (due to morphogenesis defects). Stars indicate RNAi fly lines that were tested with dicer. Stocks without the star are from the last generation and were tested without dicer.
- https://doi.org/10.7554/eLife.38853.030
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Supplementary file 7
List of VN fusion TFs that were positive in BiFC tests with VC-AbdA and tested with VC-Exd.
Green and red boxes indicate a positive or negative interaction status, respectively.
- https://doi.org/10.7554/eLife.38853.031
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Supplementary file 8
List of the 35 TFs tested as VN or CC fusion constructs with VC-AbdA or VN-AbdA, respectively.
Yellow boxes highlight the two TFs that showed opposite interaction status in the context of the two different fusion topologies (Hr83 and Ravus).
- https://doi.org/10.7554/eLife.38853.032
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Supplementary file 9
Quantification of BiFC resulting from the overlap between twi- and Ubx-Gal4 drivers in the visceral mesoderm.
- https://doi.org/10.7554/eLife.38853.033
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Transparent reporting form
- https://doi.org/10.7554/eLife.38853.034
