Multidimensional fluorescence imaging of PIP2-derived intracellular signals in directional cell movement

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Directional cell migration is important during early development, inflammatory responses to infection, wound healing, and also during tumor invasion and metastasis. Since deregulation of this process has been linked to various pathological events, it has become an active area of research for therapies. Different lines of experimental evidence suggest that various types of migratory cells share a conserved set of signals involved in cell polarization and motility. Several classes of signalling molecules, including enzymes involved in turnover and modification of phosphoinositides and components of signalling networks controlling Rho GTP-ases, play key roles in these processes. Recent studies using advanced fluorescence microscopy suggest that understanding how intracellular signals control directional cell movement is critically dependent on their dynamic organization in time and space. Fluorescence microscopy entails 'labelling' proteins of interest with fluorescent molecules ('fluorophores') such as genetically expressed fluorescent proteins that can be used to tag specific proteins in living cells. Fluorophores are 'excited' by illumination at a wavelength that they absorb and the resulting emission (fluorescence) is recorded using an imaging detector. The technique called Förster Resonant Energy transfer (FRET) works by labelling two proteins with different fluorophores or incorporating different fluorophors into single protein that changes shape in response to specific signals. The property of the two fluorophores is chosen so that the excitation spectrum on one (the 'acceptor') overlaps with the emission spectrum of the other (the 'donor') and results in the transfer of energy from the excited donor to the acceptor only if they are in close proximity. This provides a basis to measure protein/protein interactions (not just co-localisation) and to use single protein probes as biosensors. One can image FRET by observing the donor or acceptor fluorescence intensity distributions but such intensity-based FRET is often unreliable because of background noise. More reliable techniques include mapping the ratio of acceptor to donor fluorescence and fluorescence lifetime imaging (FLIM) of the donor signal. In general, fluorescence lifetime is measured by exciting fluorophores with a short pulse of light and observing how long it takes the fluorescence signal to decay away as they relax back to their ground state. Using ultrafast camera technology, it is possible to image fluorescence decays across a sample and map the fluorescence lifetime. Because FRET provides an additional route for excited donor fluorophores to lose their energy, FLIM can map where FRET is occurring by observing the resulting reduction in donor fluorescence lifetime. In a recent BBSRC project we developed a novel high-speed FLIM microscope able to 'multiplex' FRET imaging of two probes (either for protein-protein interaction or biosensors). This permits us to simultaneously map the spatiotemporal properties of two different signalling events in live cells. Here we intend to extend the approach to multiplex more simultaneous signalling events and to use FRET to focus on some of the key components controlling the directional movement of cells, in particular those associated with intracellular signals derived from a membrane phosphoinositide, PIP2. Our goal is to correlate these with other intracellular signals in the same polarized, moving cell and to analyse dynamic aspects of their timing and localisation (e.g. front and back of polarized cell). This would provide new insights into the sequence of cell signalling interactions and some underlying molecular mechanisms important for the development of therapies for various pathological events resulting from deregulation of directional cell movement. The technical innovations of FRET methodology proposed here would find wide application in biology.