MDFI of PIP2-derived intracellular signals in directional cell motility
This project is a collaboration with the group of Matilda Katan at the Institute of Cancer Research to develop and apply multidimensional fluorescence imaging and metrology techniques, particularly FLIM and FRET, to study cell signalling processes associated with cell migration.
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. Protein interactions have been widely studied by labelling each kind of protein with a different fluorophore and comparing the images at each emission wavelength. Unfortunately this “co-localisation” is limited by diffraction to a resolution of a few 100 nm - a much larger scale than the size of typical signalling proteins (~1 nm). It is possible, however, to establish when different proteins are within ~10 nm using a technique called Förster Resonant Energy transfer (FRET). This works by labelling the proteins with different fluorophores for which the excitation spectrum on one (the “acceptor”) overlaps with the emission spectrum of the other (the “donor”) and observing the transfer of energy from the excited donor to the acceptor. 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 the donor fluorescence lifetime.
In a recent BBSRC project we developed a novel high-speed FLIM microscope able to “multiplex” FRET imaging of two protein-protein interactions. 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 read out the evolution of their timing and localisation (e.g. front and back). 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.