Labeling proteins within Drosophila embryos by combining FRET reporters, position-specific genomic integration, and GAL4-reponsive expression
Chiba, A.; Deng, T.-C.; Hsieh, C.-J.; De Freitas, M.; Boulina, M.; Sharifai, N.; Samarajeewa, H.; Yanaba, T.; Baker, J. D.; Kim, M. D.; Zusman, S.; Wan, K. H.; Yu, C.; Celniker, S. E.
Show abstract
Protein interaction network (PIN) or interactome has been mapped vigorously for the entire genome. We recognize, nonetheless, that such a map could illuminate profound insights had its context been revealed. We describe a scalable protein lableling method that could re-supply natural context back to the map of protein interactome. Genetically encoded fluorescent proteins, position-specific genomic integration and GAL4-responsive expression control enable labeling proteins A, B and C each with a either an eGFP, mCherry or NirFP in specified cells of optically transparent animals such as Drosophila embryos. While following multiple proteins through development and behavior, these labels offer separable pairs of Forster resonance energy transfer between proteins A and B and proteins B and C. We test and observe FRET interactions between specific protein pairs controlling cytoskeleton, nuclear signaling and cell polarity. By using our protein labeling method, it will be possible to map protein interaction network in situ -- isPIN.
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