Journal: bioRxiv

Article Title: Temporally multiplexed imaging of dynamic signaling networks in living cells

doi: 10.1101/2022.08.22.504781

Figure Lengend Snippet: ( A ) Diagram of cell cycle indicators (known as fluorescent, ubiquitination-based cell cycle indicator 4, abbreviated FUCCI4). Dronpa, YFP, rsGreenF-E and Skylan62A were fused to cell cycle-regulated proteins Cdt1 30-120 , SLBP 18-126 , Geminin 1-110 , and histone H1.0, respectively. Analogous to the original FUCCI4, the G1-S transition is reported by the emergence of rsGreenF-E fluorescence while YFP fluorescence persists, and the S-G2 transition is marked by the fluorescence loss of YFP and stable rsGreenF-E fluorescence. Chromosome condensation, labeled by Skylan62A, indicates the M phase; finally, loss of rsGreenF-E fluorescence and the appearance of Dronpa and YFP fluorescence means the beginning of the G1 phase. ( B ) Tracking of cell cycle phase in NIH/3T3 cells via time-lapse imaging (i.e., taking brief movies at different points in time, each of which becomes a distinct datapoint in the time lapse) using TMI-based single-color FUCCI4. A mother cell dividing into two daughter cells was captured during a 11-hour imaging session, with brief movies acquired at each of the indicated times. Scale bar, 20 µm. ( C ) Fluorescence traces of Dronpa-Cdt1 30-120 (orange), rsGreenF-E-Geminin 1-110 (green), and YFP-SLBP 18-126 (Cyan) of 3 cells over their cell divisions, tracing one arbitrary daughter cell from each mother cell after M phase. Fluorescence was normalized to maximum value over the time-lapse period. Magenta bars indicate time of observation of chromosome condensation. Cell-cycle phases were assigned as depicted in A . ( D ) Diagram of TMI-based kinase translocation reporters (KTRs). A KTR contains a kinase docking site, a phospho-inhibited bipartite nuclear localization signal (bNLS) containing phosphorylation sites (P sites), a phospho-enhanced nuclear export signal (NES) containing P sites, and an rsFP. When the corresponding kinase is inactive, the KTR protein is unphosphorylated and is nuclear enriched. When the corresponding kinase is active, the KTR protein is phosphorylated and excluded from the nucleus. ( E ) Four kinase sensors were made using four green FPs: an rsFastLime-based ERK sensor, an rsGreenF-E-based JNK sensor, a Dronpa-based P38 sensor, and a Clover-based PKA sensor. NIH/3T3 cells expressing all four KTRs and H2B-TagBFP as a nuclear marker were imaged and stimulated with mouse basic fibroblast growth factor 2 (bFGF2, 20 ng/ml), which is known to drive JNK, P38, ERK, and PKA. A representative cell (out of 16 cells taken from two cell culture batches) is shown (four pseudo-channels unmixed from the green channel, and one blue channel) at the indicated time points. Scale bar, 20 µm. ( F ) Activity traces of the four kinases from the representative cell in E . R, ratio of cytoplasmic intensity to nuclear intensity. Change in fluorescence of the sensors is plotted as ΔR/R 0 (R 0 was the averaged value from t = −6 min to t= −4 min). ( G ) Averaged traces of four kinase activities recorded from NIH/3T3 cells, with 20 ng/ml bFGF2 stimulation at t = 0 min; n = 16 cells from two culture batches. Data are shown as mean ± standard error of the mean (SEM). Wilcoxon rank sum tests were run between the averaged values from t = −6 min to t = 0 min and the averaged values from t = 12 min to t = 18 min for PKA KTR and JNK KTR. Wilcoxon rank sum tests were run between the averaged values from t = −6 min to t = 0 min and the averaged values from t = 48 min to t = 54 min for ERK KTR and P38 KTR. Traces of individual cells are shown in . ( H ) Averaged traces of four kinase activities recorded from NIH/3T3 cells, with stimulation with 50 µM forskolin at t = 0 min; n = 10 cells from two culture batches. Data are shown as mean ± SEM. Wilcoxon rank sum tests were run between the averaged values from t = −6 min to t = 0 min and t = 30 min to t = 36 min for all KTRs. Traces of individual cells are shown in . Throughout the figure: *p< 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Article Snippet: Images and brief movies were acquired every 2 min. 50 μM Forskolin (Millipore Sigma), and 20 ng/ml basic fibroblast growth factor2 (bFGF2, R&D System) were used to activate kinase activities.

Techniques: Fluorescence, Labeling, Imaging, Translocation Assay, Expressing, Marker, Cell Culture, Activity Assay