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FRET has power to resolve very small distances. Yet, this potential is often left untapped in study on protein network.








to compare across proteins ..




isPIN microscopy (A) At each voxel, a confocal fluorescence microscope determines the concentration of fluorescent molecules at the three-dimensional space (x, y, z) and time (t). FRET microscope adds the association probability (%). (B) Frequency domain-modulated fluorescence lifetime imaging microscope, the type of FRET microscope we use in this work, measures mode (M) and phase (F) of frequency domain modulation. (C) Polar plot shows: (i) the fluorescence lifetime of EGFP with no FRET (from experiment with WASP alone), and (ii) the fluorescence lifetime of EGFP with partial FRET (from experiment with Cdc42 and WASp). (D) A wide range of acceptor/donor concentration ratios displays a constant lifetime of EGFP (E) Expression of fluorescently tagged proteins is kept at low constant genetic dosages by using the pan-neuronal elav-GAL4 driver. (Note: The experiments described in Figs. 2 and 3 use the cell-specific eve-GAL4 driver.) (F) Distributions of EGFP-tagged Cdc42 (top) and mCherry-tagged partner (bottom). (G) Lifetime (top) and FRET efficiency (bottom) are both absolute values specific to a given pair of interacting proteins. (H) Association probability (see (E)) is independent of the size, and thus the identity, of the partner protein.




to tag proteins reliably ..





isPIN genetics (A) EGFP and mCherry are monomeric fluorescent proeins of separate evolutionary origins. (B) Vectors for transgenes incorporate GAL4/UAS heterologous transcription and PhiC31 integrase-mediated integration. (C) Steps to create single transgene stocks. (D) Steps to combine two transgenic stocks. (E) Genetic cross for co-expresing EGFP-tagged Cdc42 and mCherry-tagged partner (Par6 or WASp) in all aCC neurons with the eve-GAL4 driver. (E) Viability test with the transgene expression.





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