Visualization of Protein Interactions in Living Drosophila Embryos by the Bimolecular Fluorescence Complementation Assay

Overview

Journal BMC Biol

Publisher Biomed Central

Specialty Biology

Date 2011 Feb 1

PMID 21276241

Citations 36

Authors

Bruno Hudry

Severine Viala

Yacine Graba

Samir Merabet

Affiliations

Soon will be listed here.

Abstract

Background: Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels.

Results: Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners.

Conclusion: Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development.

Citing Articles

Ambivalent partnership of the Drosophila posterior class Hox protein Abdominal-B with Extradenticle and Homothorax.

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Using Bimolecular Fluorescence Complementation (BiFC) with Photoactivated Localization Microscopy (PALM) to Analyze Hox/Cofactor Interactions at the Super Resolution Scale in Drosophila Salivary Glands.

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References

Popperl H, Bienz M, Studer M, Chan S, Aparicio S, Brenner S . Segmental expression of Hoxb-1 is controlled by a highly conserved autoregulatory loop dependent upon exd/pbx. Cell. 1995; 81(7):1031-42. DOI: 10.1016/s0092-8674(05)80008-x. View

Hu C, Kerppola T . Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol. 2003; 21(5):539-45. PMC: 1820765. DOI: 10.1038/nbt816. View

Takemoto K, Kuranaga E, Tonoki A, Nagai T, Miyawaki A, Miura M . Local initiation of caspase activation in Drosophila salivary gland programmed cell death in vivo. Proc Natl Acad Sci U S A. 2007; 104(33):13367-72. PMC: 1948907. DOI: 10.1073/pnas.0702733104. View

de Navas L, Foronda D, Suzanne M, Sanchez-Herrero E . A simple and efficient method to identify replacements of P-lacZ by P-Gal4 lines allows obtaining Gal4 insertions in the bithorax complex of Drosophila. Mech Dev. 2006; 123(11):860-7. DOI: 10.1016/j.mod.2006.07.010. View

Sapsford K, Berti L, Medintz I . Materials for fluorescence resonance energy transfer analysis: beyond traditional donor-acceptor combinations. Angew Chem Int Ed Engl. 2006; 45(28):4562-89. DOI: 10.1002/anie.200503873. View

Prince F, Katsuyama T, Oshima Y, Plaza S, Resendez-Perez D, Berry M . The YPWM motif links Antennapedia to the basal transcriptional machinery. Development. 2008; 135(9):1669-79. DOI: 10.1242/dev.018028. View

Taghli-Lamallem O, Gallet A, Leroy F, Malapert P, Vola C, Kerridge S . Direct interaction between Teashirt and Sex combs reduced proteins, via Tsh's acidic domain, is essential for specifying the identity of the prothorax in Drosophila. Dev Biol. 2007; 307(1):142-51. DOI: 10.1016/j.ydbio.2007.04.028. View

Ohad N, Shichrur K, Yalovsky S . The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation. Plant Physiol. 2007; 145(4):1090-9. PMC: 2151733. DOI: 10.1104/pp.107.107284. View

Fasano L, Roder L, Core N, Alexandre E, Vola C, Jacq B . The gene teashirt is required for the development of Drosophila embryonic trunk segments and encodes a protein with widely spaced zinc finger motifs. Cell. 1991; 64(1):63-79. DOI: 10.1016/0092-8674(91)90209-h. View

10.

Prasher D, Eckenrode V, Ward W, Prendergast F, Cormier M . Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992; 111(2):229-33. DOI: 10.1016/0378-1119(92)90691-h. View

11.

Azpiazu N, Morata G . Functional and regulatory interactions between Hox and extradenticle genes. Genes Dev. 1998; 12(2):261-73. PMC: 316439. DOI: 10.1101/gad.12.2.261. View

12.

Merabet S, Kambris Z, Capovilla M, Berenger H, Pradel J, Graba Y . The hexapeptide and linker regions of the AbdA Hox protein regulate its activating and repressive functions. Dev Cell. 2003; 4(5):761-8. DOI: 10.1016/s1534-5807(03)00126-6. View

13.

Kerppola T . Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu Rev Biophys. 2008; 37:465-87. PMC: 2829326. DOI: 10.1146/annurev.biophys.37.032807.125842. View

14.

Alexandre E, Graba Y, Fasano L, Gallet A, Perrin L, de Zulueta P . The Drosophila teashirt homeotic protein is a DNA-binding protein and modulo, a HOM-C regulated modifier of variegation, is a likely candidate for being a direct target gene. Mech Dev. 1996; 59(2):191-204. DOI: 10.1016/0925-4773(96)00594-1. View

15.

Gebelein B, Culi J, Ryoo H, Zhang W, Mann R . Specificity of Distalless repression and limb primordia development by abdominal Hox proteins. Dev Cell. 2002; 3(4):487-98. DOI: 10.1016/s1534-5807(02)00257-5. View

16.

Zaffran S, Kuchler A, Lee H, Frasch M . biniou (FoxF), a central component in a regulatory network controlling visceral mesoderm development and midgut morphogenesis in Drosophila. Genes Dev. 2001; 15(21):2900-15. PMC: 312807. DOI: 10.1101/gad.917101. View

17.

Peifer M, Wieschaus E . Mutations in the Drosophila gene extradenticle affect the way specific homeo domain proteins regulate segmental identity. Genes Dev. 1990; 4(7):1209-23. DOI: 10.1101/gad.4.7.1209. View

18.

Gohl C, Banovic D, Grevelhorster A, Bogdan S . WAVE forms hetero- and homo-oligomeric complexes at integrin junctions in Drosophila visualized by bimolecular fluorescence complementation. J Biol Chem. 2010; 285(51):40171-9. PMC: 3000999. DOI: 10.1074/jbc.M110.139337. View

19.

Schmid J, Birbach A . Fluorescent proteins and fluorescence resonance energy transfer (FRET) as tools in signaling research. Thromb Haemost. 2007; 97(3):378-84. View

20.

Bondos S, Tan X, Matthews K . Physical and genetic interactions link hox function with diverse transcription factors and cell signaling proteins. Mol Cell Proteomics. 2006; 5(5):824-34. DOI: 10.1074/mcp.M500256-MCP200. View