Multiplexed multiphoton fluorescence lifetime microscopy: Real time 3D imaging of protein-protein interactions by FRET

From the earliest invention of the camera, humans have been seeking to observe processes that are too fast or too complicated for the human eye and brain to determine. The first time-laspe images of a running horse allowing us to understand its motion, the moment a bullet ripped through an apple - images, freezing a moment in time so that we can examine minute details. In cellular biology our understanding of cellular function continues to evolve as we observe complex dynamic processes played out under a microscope, captured by a camera at high speed and slowly revealing its hidden intricacy. As biologists ask every more complex questions, so we must develop more sophisticated tools to rationalise the complex data that we observe. Our current understanding of protein interaction in cells is informed, principally, through the use of microscopical tools to delineate localisation and compartmentalisation of signalling events within cellular organelles such as mitochondria. Further insight can be gained regarding protein association utilising the so called Förster resonance energy transfer (FRET) technique. FRET acts as a molecular ruler, enabling us to measure the relative separation between proteins or protein-domains on the nanometer length scale. Our work has focused on the determination of protein-protein interactions by FRET and high-resolution fluorescent lifetime imaging (FLIM). Unfortunately, these advanced techniques are relatively slow in capturing cellular events so that our desire to observe real-time the processes involved in, for example cell migration (directed motion often under the action of chemical gradient) are stymied. With this project, we seek to significantly speed up the acquisition of protein-interaction data to allows us to observe cellular signalling as it happens. This can be achieved through multiplexing of our excitation and detection channels to such an extent that we envisage a 1000-fold improvement in imaging with no loss of spatial resolution in the image. This work represents the state-of-the-art in functional imaging with the opportunity to observe complex cellular events in unpreceded detail: capturing an image of a T-cell as it surveys a cancer cell, forming dynamic 3-dimensional contacts and observing the protein signalling events that drive these processes; observing the moment a cell responds to a chemical stimulus at the level of single-proteins. The technology we will develop will drammatically improve our understanding of dynamic events within cells offering insight into drug interactions in diverse applications throughout the lifesciences.

Protein interaction networks are highly regulated by spatio-temporal context within the cell. Recent methodologies for mapping these interactions using sophisticated imaging techniques have offered us considerable insight into these networks. Among the plethora of available techniques, FRET monitoring of protein-protein interactions is atractive for live cell imaging but is often stymied by limitations in dynamic range leading us to apply the most sensitive techniques such as fluorescence lifetime imaging (FLIM) to monitor both interaction distance and the fraction of molecules interacting. In this regard, we are often forced to choose trade-off resolution, for speed in an attempt to follow the temporal dynamics of evolving cellular dynamics. In order to follow fast processes in live cells we propose to develop a video rate multifocal multiphoton system for fluorescence lifetime imaging based on state-of-the-art single-photon avalanche photodiode (SPAD) arrays with on-pixel 55ps time-to-digital converter. The new SPAD array technology developed by Henderson and co-workers at the University of Edinburgh offers a huge advantage over existing fluorescence lifetime measurement tools and could present a paradigm shift in our approach to protein-interaction monitoring. We aim to exploit the advantages of this technology to, not only develop a sophisticated lifetime imaging technique, but to make it accessible to a wide variety of biological imaging problems from monitoring high-speed protein interaction dynamics to high-content screening applications using FRET readouts of cellular function or perturbation. The instrument will be characterised and exemplified in a biological context to monitor dynamic GTPase activity in cell motility using FRET biosensors. Implementation of the imaging system in a high content imaging platform will enable rapid imaging of GTPase activity in response to stimulation in a time-dependent manner.

Academic Impact: The proposal will demonstrate an eagerly sought after and novel imaging modality, providing a platform technology compatible with live cell imaging and high content screening methodologies for protein interaction screening. These developments will be of direct benefit to researchers at the life-science interface particularly the biophysics community. We seek to address a fundamental problem in biological imaging- acquisition of fluorescence lifetime imaging data with high spatio-temporal resolution at video rate. Development of the instrument at the Randall Division of cell and molecular biophysics (KCL) will allow the technology to be exploited directly by bio-imaging experts in a variety of different biological systems, including unravelling G-protein-coupled receptor conformational and signalling dynamics in cells, this has major implications for drug discovery. Ameer-Beg, Ng and Suhling are currently working within larger consortia (Joint KCL/UCL Comprehensive Cancer Imaging Centre, Optical Proteomics Network and a BBSRC funded consortium (Unravelling supra-molecular rules in signal receptor network-systems using single-molecule imaging, led by Prof Peter Parker) all of which would benefit from having this technology available. Commercial Impact: If the technology developed in this proposal is taken up by the biophotonics research community, or the pharmaceutical industry for drug discovery and high-throughput screening, it will directly benefit the commercial private sector, i.e. UK industry. The pharmaceutical industry, as a beneficiary, will find utility in the biological assays for screening potential therapeutics that have effects on chemokine receptor trafficking and the associated signalling dynamics to ultimately support the discovery, validation and development of novel therapeutics. With the emergence of molecule-targeted therapies in the clinical setting there is an increasing demand for cell-based assays which can accurately report on the changes in receptor traffic, conformation and downstream signalling through the cytoskeleton in response to treatment. Health / Societal benefits: The application of the technology to understanding cell signalling is ultimately relevant for research into diseases. The GTPase activity plays a role in cancer, AIDS, multiple sclerosis, rheumatoid arthritis, allergic disorders, asthma, psoriasis, inflammatory bowel disease and nephritis. The diversity and wide range of diseases in which these proteins are involved is of great interest to the pharmaceutical industry. The results of this proposal will thus aid drug discovery and development as a significant benefit to wellbeing and in due course will be relevant for the long-term aim of addressing in healthcare in society. Exploitation and Application: Commercially exploitable output from the proposed programme may also include establishing novel fluorescence assays based on time-resolved fluorescence spectroscopy and imaging and will be managed by KCL Business or the equivalent office at University of Edinburgh. With a background in industry, Dr Henderson is well versed in these issues. Further exploitation will occur through ongoing cell imaging research at the Randall Division of Cell & Molecular Biophysics, King's College London and via COSMIC, University of Edinburgh. Moreover, since the consortium is closely linked to one of the strongest biomedical research activities in the UK, there exist significant opportunities for the exploitation of the proposed technology. Scientific data arising from this project will be disseminated at research symposia, international conferences and peer-reviewed journals.