Figure 2: Design and validation of Fluoppi, a PPI detection system using PB1/AG tags.

(a) Schematic representation of PPI-dependent formation of fluorescent puncta. Due to the interaction between X and Y, PB1-X and AG-Y build crosslinks, resulting in the concentration of AG fluorescence (green shading). PB1 and AG are depicted as shown in Fig. 1. (b) Visualization of rapamycin-induced association between FRB and FKBP in HeLa cells co-expressing PB1-FKBP and FRB-AG transiently (left) and stably (right). To show the size of puncta, a part of the cytosol (orange box) is magnified. See also Supplementary Videos 1 and 2. The same results were obtained from approximately 50 cells in both transient and stable experiments. (c) Visualization of nutlin-3-induced dissociation of p53/MDM2 complex in two HeLa cells (cell 1 and cell 2) that transiently co-expressed PB1-p53 and AG-MDM2. See also Supplementary Video 3. The same results were obtained from approximately 50 cells in 3 independent experiments. (d) Visualization of histamine-induced oscillatory association between CaM and M13 in a HeLa cell that co-expressed M13-PB1 and CaM-KO. The image acquisition interval was 1.5 seconds. KO forms an obligate dimeric complex. The hydrophobic interface between KO monomers is indicated by a thick bar on one side. See also Supplementary Video 5. Similar oscillatory changes were observed in 4 other cells. (b–d) Domain structures of transfected constructs are illustrated (leftmost). Each domain is depicted by a rectangle. The time point of image acquisition relative to drug administration is indicated above each image. (c,d) Total fluorescence intensity of cytosolic puncta in a cell (punctum intensity, P.I.) was plotted against time (rightmost). Scale bars, 10 μm. Scale bar in magnified box (b), 1 μm. See also Supplementary Fig. 2.