Determining S6K1 localisation and interactions with mTORC1 in live cells using fluorescence lifetime imaging microscopy
The S6K1 kinase functions downstream within the mTORC1 pathway to regulate cell proliferation, aging and adiposity. Using GFP technology and advanced imaging, localisation of S6K1 has been established (47% nucleus and 53% cytoplasm). S6K1 strongly (Δτ=200 ps) interacts with the complex scaffold protein, raptor, and when the presence of the latter protein is increased, S6K1 translocates to the cytoplasm. S6K1 weakly interacts with mTOR (Δτ=100 ps) and not with Rheb which is required for the inhibitor function of rapamycin (71% vs 8.9% decrease in phospho-S6K1 without Rheb). The development of a novel biosensor (SensOR) shows phosphorylation of S6K1 occurring mainly in the cytoplasm of living cells (from τm=2.5 to 2.3 ns). In Chapter 4, AZD2014 and INK128, both pan-mTOR inhibitors, show fluorescent properties that can be used to investigate their cellular action. The fluorescence quantum yields for AZD2014 and INK128 are 0.47 and 0.33, respectively. Cellular uptake of the drugs is rapid with a half-life of 60 seconds and 42 seconds, respectively. Both drugs localise to mTORC1 related sub-cellular sites. Using cell spheroids to mimic a tumour environment, it was observed the outer spheroid layers take up AZD2014 5x faster than the inner layers. AZD2014 functions by interacting strongly with S6K1 (EFRET=18%) and Rheb (EFRET=16%) and less with mTOR and raptor (EFRET=11%). Chapter 5 investigates mass production techniques for generating sufficient quantities of S6K1 and mTORC1 proteins for future structural work. Although the baculovirus-insect cell expression system produced ~1mg of S6K1-raptor protein, impurities and degradation were present. Large quantities of the SensOR (3.7mg/ml) have been generated and purified. Solution phase studies show an open-closed SensOR conformation (from 2.7ns – 2 ns) upon the addition of ATP. Overall the research shows how FRET-FLIM technology can be usefully employed to elucidate where active drug targets must localise with regard to targeting mTOR phosphorylation.
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