High-speed imaging of FRET in live cells applied to investigate the role of PLCe in intracellular signal pathways

This multidisciplinary joint proposal between Imperial College London and the Institute of Cancer Research is to develop new technology for imaging interactions between protein molecules in live cells, to be applied to study intracellular signal pathways that are important for cancer. These protein interactions will be imaged using the fluorescence-based technique of Forster Resonant Energy transfer (FRET). Fluorescence imaging entails 'labelling' proteins of interest with fluorescent molecules (called 'fluorophores') that absorb and emit light in a characteristic manner. A recent breakthrough has been the development of genetically expressed fluorescent proteins that can be used to tag specific proteins in living cells. Conventionally the proteins of interest would be 'excited' by irradiating them with photons that can absorbed by the fluorophore labels. These would then emit light (fluorescence) and relax back to their original state. By imaging the intensity of this fluorescence, one can visualise the distribution of the fluorophores / and therefore the proteins to which they are attached. To study interactions between different proteins, one can label each kind of protein with a different fluorophore emitting at a different wavelength (i.e. different colour light). By recording images of different colours / corresponding to the distributions of each kind of protein / and superimposing them, one can see where different proteins occur in the same place, i.e. co-localisation. The problem with this technique is that the spatial resolution of the optical microscopes that are used to image living cells is limited to ~ the wavelength of the light in question (about 400-700 nm) but the proteins themselves are much smaller (~ 1-10 nm). Therefore, even if two proteins appear to occur in the same place in the fluorescence image, they can be completely independent, on a molecular scale. FRET provides a way to determine when the fluorophores are within ~ 10 nm of each other / a distance at which the proteins would be interacting. It works by observing the transfer of 'excitation energy' from one fluorophore (called the 'donor' to another (called the 'acceptor') than only occurs over this very short distance. The most straightforward way to observe FRET is to see where the donor fluorescence intensity decreases or the acceptor fluorescence intensity increases. Unfortunately this kind of intensity-based imaging is often unreliable because of background noise. The most reliable way to image FRET is by fluorescence lifetime imaging (FLIM). 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 very fast camera technology, it is possible to image fluorescence decays across a sample and obtain a value of fluorescence lifetime for each pixel in the image. Because FRET provides an additional way for excited fluorophores to lose their energy, one can determine where FRET is occurring by observing a reduction in the donor fluorescence lifetime. Unfortunately most FLIM technology is rather slow, taking several minutes to acquire a FLIM image (map of fluorescence lifetime values) and this makes it difficult to use FLIM-FRET to follow dynamics in live cells. The goal of this project is to combine novel high-speed FLIM and microscopy expertise at Imperial with the biological expertise at the ICR to develop new FLIM-FRET imaging systems. This will involve designing new molecules with appropriate donor and acceptor fluorophore labels and combining high-speed FLIM technology with novel microscope configurations. A particularly ambitious part of this proposal will be to design experiments in which we can 'multiplex FRET imaging, i.e. image two protein-protein interactions in parallel to study how different events in cells are associated in a signalling pathway.

This multidisciplinary joint proposal between Imperial College London and the Institute of Cancer Research is to develop new technology for imaging FRET of protein interactions in live cells applied to the elucidation of the role of a new member of the phospholipase C family, PLCe, in intracellular signal pathways. In particular, we first hope to clarify the interaction between PLCe and members of the Ras family of small GTP-ases, known to be implicated in a wide variety of cancerous phenotypes. The new live cell FRET imaging technology will be based on wide-field, optically-sectioned, single photon-excited, fluorescence lifetime imaging (FLIM) and related wide-field multi-spectral polarisation-resolved imaging technology. Our extensive expertise in such technologies at Imperial, including video rate FLIM, achieved by raising the S/N to unprecedented levels using new electronics and data acquisition strategies, novel tunable visible ultrafast excitation sources and in-house software tools, will be combined with the biological expertise at the ICR to design experiments and develop new fluorophore constructs. These will include GFP tagged PLCe and small GTPases labelled with mRFP to image translocation, Raichu-type FRET probes to determine specificity of the PLCe GEF domain for different GTPases and a novel FRET probe to image conformational changes in PLCe upon binding to Ras. We then aim to extend the technology to imaging multiplexed FRET interactions, correlating translocation and conformational changes with a downstream reporter of PLCe activity such as changes in intracellular calcium, using a FRET probe like Chamelon. We would explore both simultaneous (interleaved) and sequential FRET imaging, developing new FRET probe constructs to address the issues of spectral cross-talk. Linking technology development to specific biological questions should enhance our technical progress while producing useful instrumentation for a wide range of applications.