Abstract
Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation.
Highlights
Multiplexing of Förster resonant energy transfer (FRET) readouts utilising genetically expressed biosensors incorporating fluorescent proteins provides opportunities to correlate multiple signalling pathways in live cells in space and time
We report on our demonstration of time resolved fluorescence anisotropy imaging (TR-FAIM) of mCherry-Akt-PH as a quantitative homo-Förster Resonance Energy Transfer (FRET) readout for PtdIns(3,4,5)P3 and PtdIns(3,4)P2 accumulation and illustrate how this readout can be multiplexed with an enhanced cyan fluorescent protein (ECFP)-yellow fluorescent protein (YFP) FRET biosensor for calcium (TN-L15)
We developed a model for the time resolved anisotropy decay of a cluster of molecules undergoing homo-FRET was presented, relating cluster size and population fraction parameters to the parameters of a bi-exponential fit to the anisotropy decay profile
Summary
Multiplexing of Förster resonant energy transfer (FRET) readouts utilising genetically expressed biosensors incorporating fluorescent proteins provides opportunities to correlate multiple signalling pathways in live cells in space and time. Piljic et al applied spectral ratiometric imaging to FRET biosensors tagged with a mOrange-mCherry pair and a ECFP/YFP pair to monitor cytosolic calcium, membrane-bound protein kinase C (PKC) activity and annexin A4 [2]. It is possible to pair low efficiency deep red fluorophores such as mPlum with RFP donors, thereby realising a significantly greater spectral separation from ECFP-YFP This particular implementation by Grant et al, was compromised by the rapid photobleaching of TagRFP that limits its use for time course experiments. The large Stokes shift of mCherry precludes its use as a hetero-FRET donor with an acceptor of mPlum or other known far-red fluorescent proteins This motivated us to explore biosensors utilising homo-FRET readouts of mCherry that can be quantitatively analysed using time-resolved anisotropy
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