Development of novel luciferases for real-time monitoring of protein secretion.

An important way of understanding how cells and tissues function is to measure proteins that are secreted from cells, and this has traditionally been done by immunoassay techniques using antibodies. This approach has significant limitations of sensitivity in measuring very small amounts of secreted hormones or inflammatory mediators, and it is also difficult to obtain rapidly repeated measurements over time, which has hampered the development of mathematical modelling of how tissues behave in real time.
A number of organisms in nature, including fireflies, use protein molecules to generate light, a process termed bioluminescence, and the relevant genes from these animals have been identified and their structures analysed. A variety of such luciferases is now available, and some have been used in the study of gene regulation within the cell. In this application we now plan to evaluate a range of luciferases for their potential as measures of secretion. Different luciferases will be linked to proteins of biological interest such as hormones and inflammatory signalling molecules, and targeted to the secretory apparatus of the cell. Sensitive recordings of light output will be made as a measure of hormone secretion from the cell, and we will test the physical and imaging characteristics of the different proteins to assess their utility for measurement in biological fluids including blood. This is expected to lead to a new way of achieving rapid and highly sensitive assessment of secretion in real time.

This project will develop tools for the sensitive monitoring of secretion in real time using luciferase as a reporter of secreted peptide output. The long term goal is to generate a range of tools for use in a wide range of biological systems. Current methods principally rely on off-line measurement of secretion and are complicated by an inability to measure the dynamics of secretion with the time resolution required for detailed interpretation in physiological studies. Luciferases have been used extensively for monitoring gene expression and cellular localization in vivo, suggesting that modified versions may act as sensitive reporters of tissue secretion. Since there are potential issues concerning the stability of reagents and interfering factors from serum, we will characterize the properties of different luciferases and their substrates in whole blood and plasma and identify which arethe most suited to this application. To localize luciferase to the regulated secretory pathway, we will identify signal peptides which are capable of targeting luciferases to the regulated secretory pathway. The aim will be to identify generic signal peptides that can be used in a wide range of secretory cell types. Having identified the optimal luciferase and signaling peptide, we will then test the ability of fusions between these to faithfully report secretory activity in cells from a range of tissues, including the pituitary, pancreas and brain. Finally, we will test whether we can use secreted luciferase as an in vivo reporter of peptide output from tissues. The development of a form of luciferase which can act as a reporter of tissue secretion will be of value in a range of biological systems, especially those with a dynamic output which are currently challenging, particularly in small rodents such as mice where there is a limited blood volume.

The application of quantitative microscopy offers great potential for a better understanding of cell signalling and decision-making pathways. This in turn creates the opportunity for the identification of better drug targets and more efficacious modes of treatment of disease. This application is relevant to important processes in human and animal disease, for example, in inflammation and innate immunity and endocrinology. This project is important for applied research and is of relevance to healthcare and to the pharmaceutical industry.

In this respect the PI has ongoing collaborations with AstraZeneca, GSK and other companies. All the pharmaceutical companies are looking for developments in systems biology and systems medicine that prove utility for new drug target identification and validation. In this respect new reporters for biological processes are very important. We will at all stages in this project consider whether the results are of commercial value and will seek to establish specific collaborations with pharmaceutical companies when appropriate.

The major technologies being used in this project are in part based on microscopy. This is an important and currently growing area. We have close relationships with instrumentation companies and in particular with Carl Zeiss and Hamamatsu Photonics with whom MW has collaborated for 16 and 20 years respectively. This involves the loan and testing of equipment and the exchange of ideas, for new developments in microscopy and detection. Both companies (outside the present project) currently sponsor support for an annual nicroscopy training course. The companies provide speakers for the course from Germany and Japan, the loan of demonstration equipment and financial support. The current project establishes an even stronger collaboration with Carl Zeiss that will lead to close and regular interactions with the company

The use of microscopy generates movies and images that are colourful and visual. They represent an excellent resource for the development of public understanding of science. MW has given lectures at Public Understanding of Science meetings (e.g. ASE lecture in 2008). In 2006 a group from the Centre for Cell Imaging in Liverpool, led by MW and DS, presented an exhibit entitled "The Language of Cells" at the Royal Society exhibitions in London, Glasgow and at Science Day at Buckingham Palace. The whole group are very keen apply to exhibit at future Royal Society exhibitions and this project may well give us a useful theme from which to develop a new exhibit. This offers a specific opportunity and we will seek others through talking to schools and other groups. When publicity of outcomes from this project are important, we will engage with the University of Manchester Press Office to coordinate this. We have good experience of media publicity and have previously worked with funding body and University Press offices in publicising high impact publications.