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

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Description New technology for secreted luciferases identified. This is currently being developed independently by previous postdoc who has been appointed to a position at University of Edinburgh
Exploitation Route There is considerable potential for this technology for looking at cytokine and hormone secretion
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The work has assisted in a collaboration with the University of Tokyo and AIST in Japan. Two students have visited Manchester for training
First Year Of Impact 2013
Sector Education,Healthcare
Impact Types Cultural,Societal

 
Description Appointed to Pool of Experts for BBSRC Panels, 2012-2013.
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description BBSRC Bioscience for Industry Committee (single meeting for presentation)
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description BBSRC Panel: Enabling Theme 2, the Exploiting New Ways of Working Strategy Panel (ENWW)
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description BBSRC Strategic Tools and Resources Development Fund (sTRDF) Panel
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description External Reviewer for FRISYS-ZBSA (Zentrum fur biosystemanalyse), Freiberg, Germany, 26th May 2011.
Geographic Reach Asia 
Policy Influence Type Membership of a guidance committee
 
Description German Virtual Liver Scientific Advisory Board (42m euros programme )
Geographic Reach Asia 
Policy Influence Type Participation in a advisory committee
URL http://www.virtual-liver.de/wordpress/en/media/handling-biological-complexity-using-systems-approach...
 
Description Invited to attend Research Sponsors' Consultation Meeting (BBSRC &MRC) on Imaging for the biological and biomedical sciences, 14 May 2012.
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
 
Description MRC Microbiome
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description MRC Systems Immunology
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description MRC/BBSRC New Microscopy Initiative
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Member of BBSRC Research Equipment Initiative (REI) Review Panel
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of BBSRC Site visit to Imperial Centre for Systems Biology
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of BBSRC Site visit to Oxford Centre for Systems Biology
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of BBSRC Tools and Resources Development Fund Panel, Feb 2012.
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of BBSRC/EPSRC TDRI Panel
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of MRC Doctoral Training Panel
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of MRC Population and System Medicine Board, 1 April 2012 - 1 April 2016
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Membership of BBSRC Interdisciplinary Systems Biology Strategy Committee
Geographic Reach Asia 
Policy Influence Type Participation in advisory committee
 
Description Wellcome Trust/Wolfson Capital Awards in Biomedical Sciences Committee
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Carl Zeiss 
Organisation Carl Zeiss AG
Country Germany 
Sector Private 
PI Contribution We have advised Zeiss on trends in bioimaging since 1996. We have provided new data and tested prototype equipment. We have spoken at Zeiss organised meetings. We have given them an opportunity to display Zeiss equipment at our training courses. We have organised symposia that have been supported by Zeiss. We have held expert discussion meetings to review microscopy trends that have involved senior Zeiss staff
Collaborator Contribution Zeiss have made a cash contribution to training courses (received) of £16,250. Zeiss estimate of total value of in-kind staff time for collaboration, training courses and other meetings (including visits of teams from Germany) ?25,000. In addition, Zeiss have also committed over £30,000 in cash and ~£80000 in in kind staff for future training meetings and collaborative visits. Zeiss helped to design the new Systems Microscopy Centre in Manchester and made a 45% discount (value ?350k) for the purchase of equipment in 2011. Zeiss are a formal MICA partner on both Liverpool and Manchester awards from the MRC/BBSRC New Microscopy Initiative. In the award to Manchester they have made a contribution of £614,314 in staff time, development costs and equipment contribution. This involves FCS (developed during this project), luminescence fluorescence imaging, light sheet microscopy and SOFI super-resolution imaging. More recently Zeiss have made a further contribution to our new clinical single cell centre. This includes over £400k in equipment discounts and £25k in cash contribution to training and symposia. Over the years the Zeiss contributions have included them helping us with public understanding of science exhibitions where they loaned equipment, provided support for professional poster preparation and used their delivery services to transport our exhibit materials and equipment to the exhibition venues. This included an exhibition in Buckingham Palace in 2006. Zeiss have sponsored 2-3 meetings per year in Manchester. In 2016 this included a session on light sheet imaging and a session on new confocal imaging technologies. In 2017 they have sponsored an image analysis daya and will sponsor a single cell biology workshop.
Impact Annual training courses MICA collaborative MRC grant MICA collaboration on new single cell centre The relationship with Zeiss has been two way. We have been given the opportunity to be early adopters f new technology and to feedback idease for improvement. We receive very favourable deals on microscope purchases and maintenance contracts. Specific areas of successful collaboration lie in improvements to higher throughput live cell imaging using the confocal microscopes; optimisation of truly dark microscopes for quantitative luminescence imaging and the development of FCS. Multiple workshops organised (2-3 per yeat)
 
Description Hamamatsu Photonics 
Organisation PMT Hamamatsu Photonics K.K.
Country Japan 
Sector Private 
PI Contribution We have advised Hamamatsu on trends in bio-imaging for 20 years. We have provided new data and tested prototype equipment. We have given them an opportunity to display their equipment at our training courses.
Collaborator Contribution They have loaned us equipment and provided privileged discounts for almost 20 years (in kind value well over £100k). They have advised us on new emerging technology. They have assisted by providing staff for our training courses (recent in kind value calculated as £50k. They have provided £10k in cash towards training courses since 2008. A further £4k in cash and £10k has been committed to future training courses.
Impact Annual training courses