Coupled oscillators - detecting the functional consequences of signalling pathway interactions.

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

Abstract

Many of the activities of biological cells are regulated by proteins which carry signals that modify the expression of different genes at a given time, but how these signals do so is not known. Typical signals in general / such as those detected by a radio / may be encoded in terms of their amplitude (amplitude-modulated - AM) or frequency (FM), and until recently it was assumed that it was changes in the concentration (amplitude) of these signalling proteins that were important. Recently we have shown by studies in single cells that the signals in one of these pathways, the so-called NF-kappaB pathway, are oscillatory and that it is the frequency of these oscillations that seems to determine the downstream response. However, there are many other signalling pathways in a cell, and what is also unknown is the extent to which different signalling pathways are coupled to each other. Many years ago, anomalies in the orbit of Neptune led to the recognition that they must be caused by the presence of an unknown i.e. unobserved planet (Pluto), and calculations allowed astronomers both to predict the orbit of the unknown planet and thus to discover it observationally. This recognition that interactions between two dynamical systems A and B allows one to infer the presence of functional interactions solely by looking for the effects of B on A provides a novel and powerful tool that we wish to exploit here. This is because interactions between nonlinear systems oscillating respectively at A Hz and B Hz causes the production in pathway A of 'beat' frequencies of (A plus/minus nB) Hz. Since these frequencies can be measured with high precision, we seek to develop and exploit this idea for the determination of functional interactions between signalling pathways whose 'natural' frequencies of oscillation differ. This will be done both computationally (by studying a computer model of the NF-kappaB pathway, which we shall extend) and experimentally. A specific focus will be on interactions with a related and important pathway called the p53 pathway, which is modified in a large percentage of human cancers. The result will be a novel set of tools with which we can determine the interactions between signalling pathways by looking at their frequency components.

Technical Summary

We have applied single cell imaging and computational modelling to show that the NF-kappaB signalling system functions as a non-linear oscillator that controls target gene expression. We demonstrated that the excellent temporal resolution of our single cell imaging data allows detailed analysis of the characteristics of the (~100 min) oscillations of NF-kappaB proteins between the cytoplasm and nucleus. We applied a computational model, based on 64 parameters and 24 variables, to simulate the core NF-kappaB:IkappaBalpha negative feedback loop that regulates the timing and amplitude of oscillations. Despite the clear utility of this model, it lacks important components of the NF-kappaB pathway and fails to predict important aspects of our experimental data (particularly from multi-pulse re-stimulation experiments). There is also a need to consider possible stochastic as well as deterministic characteristics of the system. We will therefore improve and expand the NF-kappaB model while identifying critical parameters that are important in controlling output dynamics and function of the system. Experimentally, we have detected oscillations in other components of the NF-kappaB pathway such as in the RelB protein which can form a second oscillatory negative feedback loop with p100/p52. The RelB and RelA modules represent coupled oscillators. A major aim of this proposal is to understand from a theoretical point of view how the interaction between coupled oscillatory systems will affect output dynamics and function. We will use Fourier analysis to analyse the imaging time-course data and we will study the theoretical sensitivity of model parameters in the frequency domain. We will simulate how modulation of a hypothetical coupled oscillatory pathway would affect the NF-kappaB oscillations and relate this to the coupling of the RelA-IkappaBalpha and RelB-p100/p52 modules. Finally, we will study the interaction of NF-kappaB with the important p53-MDM2 system. We have already been able to experimentally measure oscillations in the p53-MDM2 system in single cells which occur with a 400 min period. We will now establish a computational model for the p53 system. The NF-kappaB and p53 systems are probably the two most important stress response systems in the mammalian cell that together regulate cell fate. A great deal of evidence has shown that these systems interact with each other although different mechanisms have been proposed for this cross-talk. We will study, on a theoretical and experimental basis, how these two systems with different frequencies interact with one another. These studies will act as a paradigm for the coupling between oscillatory signalling pathways and will also provide key mechanistic information on two systems that are important drug targets for inflammatory disease and cancer.

Publications

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Ankers JM (2008) Spatio-temporal protein dynamics in single living cells. in Current opinion in biotechnology

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Mullassery D (2008) Single live-cell imaging for systems biology. in Essays in biochemistry

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Paszek P (2010) Population robustness arising from cellular heterogeneity. in Proceedings of the National Academy of Sciences of the United States of America

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Sillitoe K (2007) Single-cell time-lapse imaging of the dynamic control of NF-kappaB signalling. in Biochemical Society transactions

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Spiller DG (2010) Measurement of single-cell dynamics. in Nature

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White MR (2009) Is frequency-encoding of information a major theme in cellular processes? in Cell cycle (Georgetown, Tex.)

 
Description Development of novel mathematical models to explain cell to cell variation in the NF-kB system.
Development of a new understanding of coupled systems. This developed a new theoretical framework to underpin future work on coupled systems such as NF-kB and cell cycle coupling.
Exploitation Route The tools developed have already resulted in mathematical models being used by others
Sectors Digital/Communication/Information Technologies (including Software),Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description This grant was one of the first grants on which we trained mathematicians and computer scientists. During the grant we prepared a public understanding of science exhibit that was used at the Royal Society, Glasgow and Buckingham Palace exhibitions where it was seen by many people. These resources have been used many times since. The work on the grant had a major impact in developing our systems biology strategy.
First Year Of Impact 2006
Sector Digital/Communication/Information Technologies (including Software),Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural,Societal,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 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 Dynamics and function of the NF-?B signalling system
Amount £5,072,010 (GBP)
Funding ID BB/F005938/2 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2008 
End 03/2013
 
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 Collaboration with AstraZeneca 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution We have discussed many aspects of our research with the staff from AstraZeneca, allowing them to help us to develop our research further. We also tested some candidate drugs. We have taken part in joint meetings and presented several seminars at AstraZeneca
Collaborator Contribution We had a longstanding collaboration with AstraZeneca who advise on a number of projects and have made facilities and reagents available to us. As a result of this one AstraZeneca employee was a co-author on our Science paper by Ashall et al. (19359585). This collaboration closed following the closure of the AstraZeneca Charnwood site. Recently we have resetablished this collaboration and in the next few months a senior AstraZeneca employee plans to spend time in our lab learning imaging techniques.
Impact Ashall et al (19359585)
Start Year 2006
 
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