Fig. 2

Use of splitFAST for imaging protein–protein interactions and intracellular signaling. a Use of splitFAST for imaging K-Ras/Raf1, MEK1/ERK2, and ERK2/MKP1 interactions. Representative images of cells co-expressing the indicated constructs were imaged in the presence of 10 μM HMBR (4-hydroxy-3-methylbenzylidene rhodanine) (Supplementary Fig. 8 shows control experiments). b Use of splitFAST for imaging the evolution of MEK1/ERK2 interaction upon epidermal growth factor (EGF) stimulation. HMBR-labeled HeLa cells co-expressing MEK1-NFAST and mCherry-ERK2-CFAST10 were imaged after stimulation with EGF. c Temporal evolution of splitFAST fluorescence (green), cytoplasmic mCherry fluorescence (cyan), and nuclear mCherry fluorescence (magenta) intensities (mean ± s.e.m., n = 6 cells, five experiments). Data were synchronized using the beginning of the nuclear import of mCherry-ERK2-CFAST10 as reference. d Selected frames of a representative cell (see also Supplementary Movie 3). Scale bar 10 μm. e–g Use of splitFAST for imaging of the Ca²⁺-dependent interaction of calmodulin (CaM) and the Ca²⁺-CaM interacting peptide M13. e The sensor is composed of M13-NFAST and CFAST10-CaM. f, g Temporal evolution of the intracellular fluorescence intensity for a representative HeLa cell (n = 14 cells from two experiments) treated with histamine (histamine addition is shown by the arrow) (see also Supplementary Movie 4). h–j Use of splitFAST for detecting caspase-3 activity. h Sensor design. i Selected merged frames from two-color imaging of representative HMBR-labeled cells (n = 30 cells from three experiments) expressing bFos-CFAST11 and bJun-NFAST-NLS₃-DEVDG-mCherry-NES after treatment with staurosporine (see also Supplementary Movie 5). The mCherry signal is in magenta, while the splitFAST signal is in green. Scale bar 20 μm. j Temporal evolution of the nuclear fluorescence intensity (n = 9 cells)