Dynamics and function of the NF-kappaB signalling system

Lead Research Organisation: University of Manchester
Department Name: Life 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-kB 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

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Adamson AD (2011) Novel approaches to in vitro transgenesis. in The Journal of endocrinology

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Bakstad D (2012) Quantitative measurement of single cell dynamics. in Current opinion in biotechnology

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Gonzalez-Sanchez MB (2013) Attempting to rewrite History: challenges with the analysis of histidine-phosphorylated peptides. in Biochemical Society transactions

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Gonzalez-Sanchez MB (2014) Gas-phase intermolecular phosphate transfer within a phosphohistidine phosphopeptide dimer. in International journal of mass spectrometry

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Kohlmeier F (2013) EdU induces DNA damage response and cell death in mESC in culture. in Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology

 
Description This SABR project was a joint collaboration between Liverpool (lead PI Mike White - grant BB/F005938/1), Manchester (PI Dean Jackson - grant BB/F00561X/1) and Warwick (PI David Rand - grant BB/F005814/1). All annual reports and the final report have been submitted as collaborative outputs by the lead Pi, Mike White. Please refer to his Researchfish page for relevant research metrics and outputs.
Exploitation Route Key findings related to the signalling dynamics of NF-kappaB during inflammation (see publications) and linked mathematical analyses have intimated how patterns of signalling - such as structured oscillations - can impact on target gene selection. Original work was performed in cell lines and the BAC reagents that were generated on the grant by Antony Adamson were used to establish stable cell lines that have been used extensively in the follow-on sLoLa project (lead Mike White) and also used in the generation of transgenic animals (expressing fluorescent p65 and IkappaB fusion proteins for human BACs) that have allowed signalling dynamics to be monitored and confirmed in a number of primary tissues.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description This SABR project was a joint collaboration between Liverpool (lead PI Mike White - grant BB/F005938/1), Manchester (PI Dean Jackson - grant BB/F00561X/1) and Warwick (PI David Rand - grant BB/F005814/1). All annual reports and the final report have been submitted as collaborative outputs by the lead Pi, Mike White. Please refer to his Researchfish page for relevant research metrics and outputs.
First Year Of Impact 2008
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description EC Framework 7
Amount € 3,400,000 (EUR)
Funding ID 305564-2 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 12/2012 
End 11/2017
 
Title BACs as tools for transgenesis 
Description BACs have been developed for transgenesis and use in cell lines and for generating transgenic animals 
IP Reference  
Protection Protection not required
Year Protection Granted 2014
Licensed No
Impact This protection of non-patentable IP is in progress - DAJ Antony Adamson and Mike White
 
Description Community open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact This year I manned the feedback desk for 21/2h, and engaged members of the public on many aspects of their visit and solicited views on how they felt about work in the faculty. All feedback was extremely positive and complementary.

Delivered a much clearer understanding to public about university functions.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2016
 
Description HE policy 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Informing thinking

Policy report
Year(s) Of Engagement Activity 2014
 
Description MICRA 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Community understanding and recognition of research in biomedicine

Understanding the needs of general public.
Year(s) Of Engagement Activity 2012,2013,2014
URL http://www.micra.manchester.ac.uk
 
Description Meet/ask the scientist 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Sharing information, knowledge and advice.

Helping to steer career choices.
Year(s) Of Engagement Activity 2008,2009,2011,2012,2013
URL http://www.nowgen.org.uk
 
Description Placement - pre-Uni lab experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Discussions with these students was very informative in helping them understand about the workings of university and advice on career choices

Knowledge of University life, research and ability to compose project assignment.
Year(s) Of Engagement Activity 2009,2012,2014,2016
 
Description Schools interview practice 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Feedback from students was that the experience was very helpful in building understanding of the process and confidence. 2015 2 sessions fo interviews with 10 students undertaken at Altrincham Girls School

Improving confidence and performance.
Year(s) Of Engagement Activity 2008,2009,2014,2015,2016
 
Description Summer placement - graduate students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact Encourage students to make effective career choices.

Students were informed on the value and logistics of engaging a career in academic research.
Year(s) Of Engagement Activity 2007,2009,2011,2012
 
Description Work experience - pre-Uni students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Most years we host pre-university students in the group to give them a feel of the working of university research and university life. These visit for work experience have very different durations but all serve to give students exposure to the university setting and the opportunity to explore their own feelings about their future careers. This year a gap year student Myah has been working in the lab (2 days/week for 8 weeks) to learn about biology and the value of academic research.

Generally, pre-university students have points of uncertainty regarding their career choices and even brief exposure to the research environment and academic ways of thinking is of value to their career choices.
Year(s) Of Engagement Activity 2008,2011,2012,2013,2014