Multiparameter imaging of signalling and transcription in mammalian cells

The ability to present cellular processes in the form of colour pictures and as timelapse videos, offers significant potential for presentation to lay groups. I have taken part in a number of activities that relate to public understanding of Science. These have included lectures at meetings of the British Association for the Advancement of Science (BA), local (Merseyside) and national (Radio 4) radio and television interviews. Our work has regularly attracted considerable positive press publicity locally (Merseyside newspapers), nationally (BBC and national papers) and internationally (e.g. recent interview for New York based Discover magazine). The concept of oscillating signals in cells is both novel and can be translated in simple terms for the public. I take these activities seriously, since our work can give Science and scientists a better profile. I have attended both MRC and BBSRC sessions on dealing with the Press and Public Understanding of Science.
We are generating colour videos and pictures that dramatically show important cellular events. This represents an excellent resource for further developing a programme of lay presentations. I have been approached by a local Merseyside architect, Ken Martin, regarding plans for a movie-based ?art and science? exhibit in a custom designed ?observatory? that he proposed could be housed on the waterfront in Liverpool in the Capital of Culture year, 2008. While this ambitious idea may be difficult to realise in its entirety, I would very much like to develop some aspects of this concept. In 2008, the BA annual meeting will be held in Liverpool. Significantly, the University of Liverpool Vice Chancellor, Professor Drummond Bone has just been appointed to the prestigious role of Chair of the City Culture Board (replacing Sir Bob Scott), demonstrating the increasingly close ties between the University and City in the planning of this important year.
Whenever milestones such as papers or important local meetings or events have been reached in our research, I have always worked with the University and other organisations in helping to generate good publicity. There was considerable local coverage of the formal opening of the Centre for Cell Imaging by Sir George Radda in 2003. The BBSRC have recently covered our Science paper on NF-kappaB oscillations in their BBSRC Business magazine and this and the accompanying press statement attracted international interest. I have helped several local charities with presentations to their local committees and at fund raising events.

We are applying novel multiparameter timelapse imaging technology in the Centre for Cell Imaging, to study the dynamics of signalling, transcription and cell fate. Through an MRC Career Establishment Grant, we showed that calcium signalling associated with fibroblast cell cycle entry regulates NF-kappaB-mediated activation of cyclin D1 (See et al., J. Cell Biol. 2004) We also characterized NF-kappaB as an oscillatory system (Nelson et al., Science, 2004). These discoveries provide novel insights into the mechanism of NF-kappaB control of transcription and cell fate, as well as its interactions with other signalling pathways. Our previous published work described the dynamics of two NF-kappaB proteins, RelA and IkappaBalpha, fused to fluorescent proteins. The NF-kappaB network involves more than 10 further proteins that form at least 4 negative feedback loops, with the potential to regulate the period and amplitude of NF-kappaB oscillations and thus to differentially regulate transcription. At least 3 of these (RelB, p100/p52 and p105/p50) give rise to distinct NF-kappaB signalling kinetics. We propose to define the integrated roles of the family of NF-kappaB proteins in cell lines and clinically relevant primary models to decipher their contribution to gene expression pathways leading to cell survival or cell division. Together with cell imaging, we will apply ChIP assays (with Mahadevan and Metivier) to study the kinetics of NF-kappaB binding to chromatin and transcription complex assembly. We will also study NF-kappaB binding in living cells (with Mancini).
Aims that we will investigate include: 1) The roles of NF-kappaB oscillations for transcription control. 2) The contribution of each NF-kappaB family member (A20 with Evans) to differential transcription. 3) The role of essential NF-kappaB protein modifications (mainly RelA) for oscillation dynamics and function? 4) Investigation of the mechanisms and consequences of cross-talk with other signalling pathways (calcium, glucocorticoid receptor (with Ray) and p53) for the regulation of cell fate. 5) How NF-kappaB responses vary in primary cells and pathological situations (neuroblastoma, with Losty and H. pylori-induced gastric cancer, with Dockray and Varro). This work will benefit from collaborations with scientists with complementary expertise (including mathematical modelling (Kell) and pharmaceutical drugs and assays (Unitt, AstraZeneca).
The NF-kappaB pathway is now one of the most characterised examples of a protein-based signalling system that involves spatio-temporal oscillations that intimately control function. The development of a detailed dynamic analysis of this network promises to promote a paradigm-shift in the way that scientists think about signalling processes and cellular responses.