Douglas J. Kelly

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.

FOXO3a is a forkhead transcription factor that regulates a multitude of important cellular processes, including proliferation, apoptosis, differentiation, and metabolism. Doxorubicin treatment of MCF-7 breast carcinoma cells results in FOXO3a nuclear relocation and the induction of the stress-activated kinase p38 MAPK. Here, we studied the potential regulation of FOXO3a by p38 in response to doxorubicin. Co-immunoprecipitation studies in MCF-7 cells demonstrated a direct interaction between p38 and FOXO3a. We also showed that p38 can bind and phosphorylate a recombinant FOXO3a directly in vitro. HPLC-coupled phosphopeptide mapping and mass spectrometric analyses identified serine 7 as a major site for p38 phosphorylation. Using a phosphorylated Ser-7 FOXO3a antibody, we demonstrated that FOXO3a is phosphorylated on Ser-7 in response to doxorubicin. Immunofluorescence staining studies showed that upon doxorubicin treatment, the wild-type FOXO3a relocalized to the nucleus, whereas the phosphorylation-defective FOXO3a (Ala-7) mutant remained largely in the cytoplasm. Treatment with SB202190 also inhibits the doxorubicin-induced FOXO3a Ser-7 phosphorylation and nuclear accumulation in MCF-7 cells. In addition, doxorubicin caused the nuclear translocation of FOXO3a in wild-type but not p38-depleted mouse fibroblasts. Together, our results suggest that p38 phosphorylation of FOXO3a on Ser-7 is essential for its nuclear relocalization in response to doxorubicin.

We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100's to 1000's of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. KD values broadly agree with published biochemical measurements.

The forkhead box transcription factor FOXM1 is an essential effector of G2/M-phase transition, mitosis and the DNA damage response. As such, it is frequently deregulated during tumorigenesis. Here we report that FOXM1 is dynamically modified by SUMO1 but not by SUMO2/3 at multiple sites. We show that FOXM1 SUMOylation is enhanced in MCF-7 breast cancer cells in response to treatment with epirubicin and mitotic inhibitors. Mutation of five consensus conjugation motifs yielded a SUMOylation-deficient mutant FOXM1. Conversely, fusion of the E2 ligase Ubc9 to FOXM1 generated an auto-SUMOylating mutant (FOXM1-Ubc9). Analysis of wild-type FOXM1 and mutants revealed that SUMOylation inhibits FOXM1 activity, promotes translocation to the cytoplasm and enhances APC/Cdh1-mediated ubiquitination and degradation. Further, expression of the SUMOylation-deficient mutant enhanced cell proliferation compared with wild-type FOXM1, whereas the FOXM1-Ubc9 fusion protein resulted in persistent cyclin B1 expression and slowed the time from mitotic entry to exit. In summary, our findings suggest that SUMOylation attenuates FOXM1 activity and causes mitotic delay in cytotoxic drug response.

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated.