Dynamics and function of the NF-kappaB signalling system

Lead Research Organisation: University of Liverpool
Department Name: Sch of Biological Sciences

Abstract

A major challenge in biology is to understand how cells recognize external signals and give appropriate responses. Now that the sequence of the human genome is complete, it is important to assign functions to each gene and to identify the corresponding proteins that control key cellular functions. White and colleagues pioneered the development of microscopy-based methods for the visualization and timelapse measurement of biological processes in single living cells. We have used natural light-emitting proteins from fireflies, jelly fish and fluorescent corals. Synthesis (expression) of these proteins causes mammalian cells to become luminescent (light emitting in the dark) or fluorescent (change the colour of light). By placing the gene that codes for a luminescent protein next to a promoter that controls a gene of interest, we can use luminescence from living cells as a way of measuring when the gene of interest is normally switched on and off. Fluorescent proteins have also been used to genetically label proteins of interest, so that the movement of the protein can be visualized in a living cell. White and colleagues previously used timelapse fluorescence and luminescence microscopy coupled to computer simulations to investigate cell decision making. We discovered that a set of important signalling proteins, called NF-kappaB, move repeatedly into and out of the nucleus of the cell, suggesting that cells may use proteins as timers to encode complex messages (like Morse Code). This was a surprise since the original NF-kappaB protein, p65, was discovered 20 years ago and was thought to act as a simple switch that moves into the nucleus once to activate genes. Only timelapse measurements in single living cells were able to see this. The NF-kappaB system is widely recognised as crucial to the control of important cellular processes including both cell division and cell death. It is implicated as being involved in a variety of diseases, such as cancer and inflammatory disease. We will now develop a substantial systems biology project to study all of the components of this complex system. While the previous work has provided major insights, we now need a far broader range of integrated experimental tools to study it. Also the use of mathematical models to make computer predictions will be critical to help us to visualize how this system works. We will make accurate measurements of the (much larger) set of proteins that are involved in NF-kappaB signalling and the genes that are controlled by these signals. The (very experienced) project team includes bioinformaticians, cell biologists, computer scientists, mathematicians, molecular biologists, microscopists and protein chemists. The project will be managed in a structured and organized way, so that the mathematical modelling can be used to predict and design the biological experiments. A central team of experimental officers will be responsible for coordinating the experiments, data and model storage and communication of information between team members. We will study the numbers of molecules of each of the NF-kappaB proteins in the cell, their stability, chemical states and interactions with each other and with other proteins. We will also study in detail which genes that they bind to and control. We will also aim to understand how single protein molecules acting at single genes can act to control decisions of cell life and death. This multidisciplinary approach is essential in order to understand this complex system. A further aim of the project is to provide training for post-docs and students. In this respect, we will benefit from sponsorship of training courses and symposia by the instrumentation companies Carl Zeiss, Hamamatsu Photonics, Coherent and Nano Imaging Devices. The project will also benefit from ongoing collaborations with Genetix and AstraZeneca

Technical Summary

We will develop an integrated systems biology programme to analyse the dynamic and physiological function of the NF-kappaB signalling system. We previously applied iterative real-time imaging and mathematical modelling approaches to show that the NF-kappaB system is oscillatory and uses delayed negative feedback to direct nuclear to cytoplasmic cycling of transcription factor(s) that regulate gene expression. Our recent work has made clear how little is currently understood about even the core parts of the NF-kappaB system and only included a small subset of the NF-?B proteins and feedback loops. We will develop and apply a set of quantitative experimental tools coupled to an intensive theoretical analysis to properly analyse the dynamic function of the system. A key question is how cells achieve appropriate cell fate decisions in response to time-varying signals. Our team includes the expertise to measure and simulate the important processes involved in the core NF-?B network and is supported by leading technology companies.. The experimental work (involving network perturbations) will integrate dynamic cell and single molecule imaging, quantitative proteomics (for measurement of absolute protein and phosphoprotein levels and rates of turnover), chromatin immunoprecipitation (ChIP) analysis (for the dynamics of NF-kappaB binding to target promoters) and RT-PCR and DNA microarray analysis (for measurement of endogenous gene expression). The theoretical work will develop: 1) new data analysis tools to interpret and direct experimental strategy, 2) deterministic and 3) stochastic mathematical models of the system. The computer simulations will develop new experimentally testable hypotheses. Our goal is complete understanding of this complex and non-linear system. We will determine how the set of complex feedback loops controls NF-kappaB dynamics and controls downstream gene expression in the nucleus and how it operates at the single molecule/gene level.

Publications

10 25 50

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/F005938/1 01/05/2008 30/09/2010 £3,226,314
BB/F005938/2 Transfer BB/F005938/1 01/10/2010 30/04/2014 £1,658,302
 
Description We have performed a thorough characterisation of the way in which the NF-kB signalling system responds to inflammation and stress. This has involved biochemical. cell imaging and mathematical analysis of the system.
1. We found that pulsatile signalling can drive NF-kB translocation at different frequencies. This can alter the pattern of gene expression.
2. We showed that oscillations in NF-kB between the cytoplasm and the nucleus occur in multiple different cell lines and in primary cells from transgenic animals. We developed a new genetic reporter system to achieve this
3. We showed that heterogeneity in the timing of oscillations between cells may be an engineered feature of the system that is important to allow accurate control of inflammation at the tissue level.
4. We discovered a set of new phosphorylation sites on the protein p65. Some of these control dimerization and others control localisation and gene expression.
5. We discovered that temperature controls NF-kB oscillation timing. Subsequent analysis of gene expression implicated the feedback inhibitor A20 in this process. This implies that the inflammatory response may change with fever.
6. The anti-inflammatory drugs aspirin and diclofenac alter the timing of NF-kB translocation.
This work is onging through a new programme which is concentrating on studies in primary cells and tissues.
Exploitation Route the project has led to substantial collaborations with instrumentation companies.
Mathematical models developed in the project are being widely used and developed by other labs
Transgenic reporter mice developed at the end of the project show considerable promise as a model system to study inflammation in a variety of primary cells and tissues. The genetic bacterial artificial chromosome approach has been used by other labs and is providing an invaluable tool for studying a variety of other signalling processes.
There is ongoing collaboration with two major pharmaceutical companies
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description We trained four PhD students and a set og biological and mathematical postdocs in systems biology. This included specific weekly training. This led in part to the suggestion for online systems biology training via the BBSRC Interdisciplinary Systems Biology Strategy Committee. With Zeiss and Hamamatsu we ran microscopy training courses that were available to outside scientists. We worked with Zeiss to improve the automated imaging software. These and other outputs were more fully described in our SABR final report
First Year Of Impact 2008
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic,Policy & public services

 
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 LoLa oanel
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
URL https://bbsrc.ukri.org/funding/filter/lola/
 
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 (W Muller, MW, D Jackson, S Schreiber, T Hofer, D Rand, C Troutwein, J Rhodes, M Pierik, N Schpigel, VM dos Santos, A Kel, L Ferguson, B Fuchs). Systems medicine of chronic inflammatory bowel disease.
Amount £2,583,208 (GBP)
Funding ID 305564 
Organisation Systems Medicine of Chronic Inflammatory Bowel Disease 
Sector Public
Country European Union (EU)
Start 12/2012 
End 11/2017
 
Description A "Molecular Imaging (FLIM/FCS) toolbox" to investigate molecular interactions and activation in super-resolution and widefield mode
Amount £255,000 (GBP)
Funding ID 202923/Z/16/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2016 
End 10/2020
 
Description An upright confocal microscope for multidisciplinary research
Amount £282,781 (GBP)
Funding ID BB/R014361/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 04/2019
 
Description BBSRC David Phillips Fellowship to Pawel Paszek
Amount £946,737 (GBP)
Funding ID BB/I017976/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2011 
End 09/2016
 
Description Capital clinical infrastructure for single cell genomics
Amount £4,900,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 04/2015 
End 03/2020
 
Description Imaging of cellular dynamics from single molecules to tissues.
Amount £1,273,491 (GBP)
Funding ID MR/K015885/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 04/2013 
End 04/2017
 
Description MRC Clinical Research Training Fellowship
Amount £218,114 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2007 
End 01/2009
 
Description Strategic Lola
Amount £4,160,524 (GBP)
Funding ID BB/K003097/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2013 
End 04/2018
 
Description Temporal manipulation of genetic circuits in single cells
Amount £122,019 (GBP)
Funding ID BB/P027040/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2017 
End 09/2018
 
Description Wellcome Trust 4-year PhD Programme in Quantitative and Biophysical Biology
Amount £2,555,000 (GBP)
Funding ID 108867/B/15/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2017 
End 01/2023
 
Title Bacterial Artificial Chromosomes 
Description We have made bacterial artificial chromosomes expressing most of the key NF-kappaB proteins fused to fluorescent proteins. Cell lines have been made from several of these. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact These have been used to make stable cell lines and more recently IkBalpha-EGFP and p65-DsRed expressing transgenic mouse lines. Initial data suggests that these will be extremely powerful research tools. 
 
Title Development of fluorescence correlation spectroscopy for protein quantification and interaction measurement 
Description Tool based on confocal microscopy for measurement of absolute fluorescent protein concentration and measurement of protein dissociation constants in single living cells. They method involves a combination of experimental measurement and mathematical analysis of the data. Further development to consider use with light sheet microscope in collaboration with Zeiss. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact Papers published. Collaboration with Carl Zeiss. 
 
Title Mathematical models of the NF-kappaB signalling pathway 
Description Models that have bee n published have been made available online. see http://www.liv.ac.uk/bio/research/groups/cci/ 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact There has been substantial interest in these models. 
 
Title NF-kappaB protein expression plasmids 
Description Plasmids which have been published have been made abailable to other researchers. We have made plasmids and cell lines available when published. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact We now regularly receive a large number of requests for plasmids and some requests for a cell line that relates to this work. These plasmids have been used for the construction of bacterial artificial chromosomes (which are as yet unpublished). These are now being used to construct transgenic animals 
 
Title Reporter transfenic mice 
Description We have used a series of Bacterial artificial chromosome reporter constructs to generate transgenic mice expressing human fusion proteins for key transcription factors. Initial examples are RelA/p65 and IkBa in the NF-kB system. The cells from these animals have proved extremely useful and show dramatic dynamics in NF-kB translocation and target gene expression. We are also in the process of generating a set of reporter mice for other key pathways (e.g. NRF2 and p53). These are likely to be a very powerful resource. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact First evidence of NF-kB oscillations in vivo and evidence that different cells in different contexts show differing dynamics. We aim to develop a plan to share these animal lines with interested groups 
 
Title Mathematical models of NF-kB and inked systems 
Description ODE and stochastic models of the NF-kB system and other modules that couple with it 
Type Of Material Computer model/algorithm 
Provided To Others? Yes  
Impact Multiple citations with other papers being based on our models 
 
Description Collaboration with University of Liverpool 
Organisation University of Liverpool
Department Institute of Integrative Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Contribution of data and intellectual input on shared BBSRC grant. Collaboration on student training.
Collaborator Contribution Contribution of data and intellectual input on shared BBSRC grant. Collaboration on student training. (This was a continuing collaboration following move of M. White from Liverpool to Manchester)
Impact Multi-disciplinary - including mathematics, translational medicine and proteomics.
Start Year 2010
 
Description Collaboration with University of Manchester 
Organisation University of Manchester
Department Manchester Interdisciplinary Biocentre
Country United Kingdom 
Sector Academic/University 
PI Contribution We have contributed ideas and data and we have recently transferred expertise for BAC expression technology to the Manchester group,
Collaborator Contribution Several staff from Manchester University collaborated in our application to BBSRC for a SABR initiative grant. Some of these staff had collaborated throughout the present MRC grant while others were new collaborators. Their contibutions were in nuclear structure and function, mathematics and databasing. (This collaboration 'ended' following the move of M. White from Liverpool to Manchester)
Impact This collaboration led to the award of the £5m BBSRC SABR grant between Warwick, Liverpool and Manchester. The collaboration supported the Science paper by Ashall et al., (19359585) This ceased to be an external collaboration when M. White moved to Manchester University
 
Description Collaboration with University of Warwick 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution We have shared data and made visits to Warwick to discuss the collaboration.
Collaborator Contribution We have established a strong systems biology collaboration with the Systems Biology Department in Warwick (particularly with Prof. D. Rand). They have become our principal mathematics collaboration and contributed very significantly to our recent Science paper (Ashall et al.,) and other papers submitted or in preparation. This collaboration included the Warwick group (T. Brettschneider) using their own funds to assist us in the development of cell imaging programs through their employment of a post-doc. We also have collaborated over student training.
Impact This multidisciplinary collaboration led to the award of the £5m BBSRC SABR grant between warwick, Liverpool and Manchester. This directly arose from this collaboration with the Warwick group assisting with the predictive modelling work. The collaboration supported the Science paper by Ashall et al., (19359585) In 2010 through more distantly related work, a £1.4m Wellcome trust Programme grant was awarded to Manchester and Warwick. In 2013 a further directly related £3.6m BBSRC sLOLA grant was awarded to Manchester and Warwick.
Start Year 2007
 
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
 
Description School Visit, Altrincham Girls School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Dean Jackson - Advice and practice interviews for students
Year(s) Of Engagement Activity 2016
 
Description School visit (Liverpool Life Sciences, University Technical College) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Liverpool Life Sciences UTC is the first school in the UK specialising in Science and Health Care for 14 to 19 year olds. Talk at Liverpool Life Sciences UTC conference on "How Organisms Age"
Year(s) Of Engagement Activity 2016
 
Description School visit, (Manchester Grammar School) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Dean Jackson Feb 2017, Science Fair judge for student projects
Year(s) Of Engagement Activity 2017
 
Description school visit (Xaverian College) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Dean Jackson - STEM ambassador
Year(s) Of Engagement Activity 2016