TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells

摘要

a Schematic of split HaloTag design. The structure was modified from DhaA (1BN6). The scissor represents the split site and the green and red colors represent the two split halves of HaloTag. The displayed split site is P58 in HaloTag (P68 in 1BN6). b Fluorescent retention assay for the screening of potential split sites. Split HaloTag at different positions was fused to two interaction protein fragments, β-Fos and β-Jun, respectively. Functional HaloTag will be reconstituted when β-Fos and β-Jun interact if the split site is proper. Membrane-permeable HaloTag ligand dye was added to the live cells. The fluorescence will remain after washing. Mutation of dimerization domain of β-Fos was used as a control. c Violin plot of the retention fluorescence intensity of split HaloTag at different split sites. The red dots are the average intensity of HaTagN-β-Jun+β-Fos-HaloTagC while the blue dots are the average intensity of HaTagN-β-Jun+β-Fos (ΔZip)-HaloTagC. The size of the red dot represents the ratio of HaTagN-β-Jun+β-Fos-HaloTagC to HaTagN-β-Jun+β-Fos (ΔZip)-HaloTagC. d Fluorescence images of reconstituted split HaloTag at P58 and G261 split sites. The images were displayed at the same intensity range for comparison. The cell nuclei were stained by DAPI. Scale bars, 5 μm. e Typical florescence image of one cell nucleus expressing reconstituted split HaloTag (HaloTag N58-β-Jun and β-Fos-HaloTag C58). The cells were fixed immediately after the incubation of JF549 dye for 15 min. The white dot line delineated the cell nucleus. Scale bar, 2 μm. f Kymograph of single-molecule fluorescent trajectory of split HaloTag (from e). A long zigzag line was drawn in the nucleus (not shown in e) and fluorescence intensity of the line scan was displayed over time. g One typical intensity profile showing single-step photobleaching of the TagBiFC signal (from e).