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

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.

Building on our ongoing collaboration, this multidisciplinary joint proposal aims to study cell signalling, in live cells and in vitro, using novel fluorescence imaging technology to measure FRET, particularly using biosensors. In our previous BBSRC funded project (BB/E003621/1) we successfully developed custom FRET constructs and robust new multiplexed FRET imaging technology to independently read out two signalling events simultaneously. This was complemented by the development of a multidimensional fluorometer for cuvette-based studies, including FRET, at Imperial (BB/E000495/1). We now wish to apply this new technology to study the signalling networks controlling directional cell movement. We plan to focus on intracellular signals derived from PIP2, which we have extensively studied at the ICR. Our multiplexed FRET methodologies give us a unique capability to tackle one of the main challenges, i.e. to correlate different dynamic signalling events. First, we will determine which candidate molecules (PLC isoforms) involved in intracellular signalling from PIP2 are critical to regulation of directional movement of fibroblasts towards platelet-derived growth factor. We will then correlate signalling events generated by PLC and PIP2-derived signals with activation of other components involved in cell movement, particularly Rho GTP-ases, and will test the links between them. For this we will make FRET constructs from known biosensors and refine and apply fluorescence instrumentation utilising spectral, polarisation and time-resolved measurements. Multiplexing FRET biosensors will permit us to study the timing and localisation (front and back of a polarized, moving cell) of specific signalling events under the same conditions, within the same cell. We will further determine the molecular mechanism of PLC activation by Rac, a molecule with an established role in cell movement, through in vitro FRET studies.

The biological questions we propose to address have direct relevance to understanding various diseases including cancer, with clear impact on the development of more effective therapies, thereby enhancing the quality of life for patients and providing significant commercial opportunities. In general, the outcomes of this project will be directly relevant to scientists using fluorescence - in almost any field of science - since FLIM provides a robust tool for molecular imaging, contrasting different fluorophores and different fluorophore environments. Multiplexed FRET is especially useful to life scientists wishing to study cell signalling networks. This is a vital area for the understanding of disease and the development of more effective therapies. The specific results related to cell movement would directly impact tumour invasion and metastasis as targets for therapies. Longer term beneficiaries therefore include patients suffering from diseases, clinicians and the taxpayer, who would benefit from the reduced healthcare costs associated with more effective therapies. The FRET based biosensors that we will develop could be applied in a wide range of disease related fields including cancer. They could be used by scientists in universities, research institutes and industry. In the medium term, the biosensors and fluorescence-based technology could be applicable to many fluorescence assays and so would be applicable to drug discovery, e.g. using High Content Analysis and other approaches. The outcomes of this project could therefore be of significant commercial interest to pharmaceutical companies and the drug discovery industry in general - including, assay developers, instrumentation developers and drug screening companies. The pharmaceutical sector is important to the UK economy and the ability to study multiple events in cell signalling networks could enhance the success of screening campaigns and the efficiency of the drug discovery pipeline. Developing even one successful drug brings enormous financial benefits and the clinical benefits directly impact the quality of life for patients. If the efficiency of the drug discovery process can be improved, this will reduce the cost of drug discovery and therefore the cost of successful drugs. It could also reduce the number of animals required for testing drugs. Besides giant pharma, there are many smaller companies and SME's in the UK who innovate assay and instrumentation development for drug discovery. Access to a range of proven biosensors could create new opportunities for assay developers and the demonstrated success of multiplexed FRET would create new commercial opportunities for instrumentation manufacturers who could upgrade their microscopes and plate readers. Successful outcomes of this project would also drive the development of software tools since there are many challenges in the data analysis of multidimensional time lapse fluorescence imaging and this would create employment and commercial opportunities in the software sector. We anticipate that this project could immediately lead to new drug targets and the impact on assay developers and instrumentation manufacturers could be significant within 5 years. The researchers working on our project would develop valuable skills in molecular biology, imaging, software development, instrument design and data analysis. This could lead to employment in the pharmaceutical or other industrial sector, academic or clinical research or in consulting or policy development in the public or private sectors. For commercial exploitation, the ICR's dedicated department for technology transfer and exploitation will liaise with Imperial Innovations. We note that Matilda Katan has an established link with Cancer Research Technology Limited (CRT Ltd) with the aim to implement new therapies for direct patient benefit and Mark Neil is a cofounder of a successful microscope spin-out company (Aurox Ltd).