Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in Caenorhabditis elegans.
Protein interactions are essential components of signal transduction in cells. With the progress in genome-wide yeast two hybrid screens and proteomics analyses, many protein interaction networks have been generated. These analyses have identified hundreds and thousands of interactions in cells and organisms, creating a challenge for further validation under physiological conditions. The bimolecular fluorescence complementation (BiFC) assay is such an assay that meets this need. The BiFC assay is based on the principle of protein fragment complementation, in which two non-fluorescent fragments derived from a fluorescent protein are fused to a pair of interacting partners. When the two partners interact, the two non-fluorescent fragments are brought into proximity and an intact fluorescent protein is reconstituted. Hence, the reconstituted fluorescent signals reflect the interaction of two proteins under study. Over the past six years, the BiFC assay has been used for visualization of protein interactions in living cells and organisms, including our application of the BiFC assay to the transparent nematode Caenorhabditis elegans. We have demonstrated that BiFC analysis in C. elegans provides a direct means to identify and validate protein interactions in living worms and allows visualization of temporal and spatial interactions. Here, we provide a guideline for the implementation of BiFC analysis in living worms and discuss the factors that are critical for BiFC analysis.
Hiatt
Susan M
SM
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue Cancer Center, Purdue University, 575 Stadium Mall Drive, RHPH224D, West Lafayette, Indiana, IN 47907-2091, USA.
Shyu
Y John
YJ
Duren
Holli M
HM
Hu
Chang-Deng
CD
D000818:Animals; D001681:Biological Assay / Q000379:methods; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000032:analysis / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D005822:Genetic Vectors; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism / Q000528:radiation effects; D014162:Transfection; D020798:Two-Hybrid System Techniques
D029742:Caenorhabditis elegans Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins