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Research @ the University of Liverpool

Winter 2010 - Movements in space and time

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Scientists at the University of Liverpool are developing cutting-edge tools to visualise cellular processes at the nanoscale. Understanding the exact movements of proteins and other molecules within cells could have wide-reaching applications in biomedicine.

Over the past 15 years, live cell imaging has really taken off. The development of luminescent and fluorescent tools, such as firefly luciferase and green fluorescent protein (GFP), has enabled researchers to watch processes that were previously invisible, such as the unexpected dynamics of the movement of signalling molecules inside cells and the surprisingly dynamic pulses of activity from many genes.

Mapping the roles of the thousands of different proteins that control important chemical processes is now central to modern bioscience.

Using real-time fluorescence imaging, Professor Mike White, Director of the University’s Centre for Cell Imaging, is leading research on NF-kappaB, a protein that, amongst other things, coordinates immune responses to infection. By tracking the movement of NF-kappaB within single cells, White’s team have discovered new and unexpected features of a sophisticated communication system that coordinates the setting up and maintenance of inflammation within the body by broadcasting a signal to surrounding cells.

"The localisation of NF-kappaB in cells forms a wave-like pattern, which oscillates over time," White explains.

Turn on, tune in

But while real-time imaging itself is revealing dynamism within the system, White’s team is increasingly reliant on a multidisciplinary approach to understand how it arises and what functions it may have. In a joint project involving biologists and mathematicians from the Universities of Liverpool, Manchester and Warwick, White was able to show that different cell processes are switched on once they pick up the frequency of peaks and troughs in the NF-kappaB signal, just like tuning into an FM radio signal.

Importantly, this was the first demonstration, within a protein pathway, that it is the frequency of the signal and not the amplitude – the amount of protein – which results in different cellular responses. This paradigm shift in thinking could have important impacts on our understanding of how biological processes are controlled.

"Wherever we look we see surprising dynamics in cellular processes," says White.

Lots of systems have now been shown to oscillate, such as calcium signalling and more recently other protein systems such as the p53, STAT, Notch, Wnt and ERK signalling systems. The key now is to understand the functional significance of these oscillations.

"Like different sized cogs in a watch, oscillating systems may allow cells to make robust decisions and avoid making mistakes". If you were only measuring amplitude it would be incredibly difficult to get such a dynamic range of responses.

And while this explanation is intuitively understood by physicists, White believes that large sections of the biological sciences research community are probably still to pick up on the significance of this mechanism.

White’s next step is to look at the sources of heterogeneity in cellular tissues and how this may arise. He believes that this may have specific biological advantages

A key part of this work is the building and testing of mathematical models that allow simulation of the multiple feedback loops that lead to oscillations and how and why cells often seem to be out-of-phase with their neighbours.

Data driven modelling

In an approach that combines cell imaging with proteomics, Prof. White and colleagues are working with protein chemist Professor Rob Beynon in the School of Veterinary Preclinical Sciences to develop new data analysis tools to interpret and direct experimental strategy, including how cells make decisions in response to time-varying signals.

"It currently takes us weeks to analyse oscillations," says White. "We need accurate data on how many molecules of NF-kappaB there are in the system, and the rates of protein turnover. This is important to allow us to relate our imaging data to the numbers of molecules of each protein".

"One of the questions we need to answer is whether the number of molecules of GFP-tagged protein are comparable to the real system," says Beynon. "There is an ever present danger that your protein may not function correctly with a large fluorescent molecule stuck to it, or it may be produced in abnormally high amounts, swamping other cellular processes".

Everything happens for a reason

Cell ‘fate’ is not something that happens by chance. Signalling molecules such as transcription factors play a key role in cell decision-making processes: to divide or not to divide; to differentiate or not differentiate; to live or to die. Understanding cell fate has important implications for our understanding of how healthy organisms develop, as well as when things go wrong.

BBSRC David Phillips Research Fellow Violaine Sée is using single cell imaging to examine factors that influence cell fate, such as the link between the oxygen environment of the cell and the move from a normal, physiological state to tumour formation.

In the body, cells are typically exposed to around 3-5% oxygen – a lot lower than atmospheric levels – and oxygen conditions down to about 1% are common in the centre of solid tumours, something that could contribute to tumour aggressiveness.

"Cell cultures are usually performed under atmospheric conditions; we want to look at how physiological conditions affect cell processes," explains Sée. "We’re looking at how cells adapt to low oxygen conditions".

Sée’s team developed a method to track the movement of a protein called hypoxia inducible factor (HIF), which switches on the cell’s stress response to low oxygen.

Cells continuously produce and degrade HIF so it is not normally detectable. But when oxygen concentration goes down, HIF accumulates in the cell nucleus, triggering a cascade of genetic responses, and then decreases slowly over 48 hours – at least that’s what has been observed in whole cell populations.

"What’s interesting," says Sée, "is when we look at a single cell, we see a transient accumulation of HIF – a sharp peak up and down over 4-6 hours. What is more, intriguing is that each cell seems to be doing this asynchronously – this heterogeneity is new and very exciting."

"We have found that the accumulation of HIF is dependent on the cell’s position in the cell cycle – whether it is ‘resting’ or actively replicating and dividing – if HIF is accumulating when the cells are not ready, it could have serious consequences. This discovery was only made possible by looking at single cell data."

Sée is working with a mathematician, Dr Rachel Bearon, to predict which HIF patterns are the most important to look at experimentally. Ultimately, they hope to use the single cell data to develop a multi-scale tissue model to evaluate potential new anti-cancer drugs, including those that target HIF.

Beyond GFP

Inspired by naturally occurring biomolecules, another BBSRC David Phillips Fellow, Dr Raphaël Lévy has designed nanoparticles as probes that can be visualised over many hours and do not have the drawback of photobleaching so often encountered with fluorescent labelling. Ultimately, Lévy aims to use these particles to watch single molecules as they move around the cell.

Because gold nanoparticles have a very simple chemistry, Lévy is able to vary the biomolecules used to coat them and produce particles with different properties. Using different peptide sequences as a coating layer, he is able to regulate uptake by living cells and limit non-specific interactions.

Lévy has built one of only two photothermal microscopes in the world, and the first to be specifically designed for imaging nanoparticles in living cells. The microscope works by firing lasers, one pixel at a time, at cells containing gold nanoparticles. These particles take in visible light, giving out heat, and this change in temperature is detected, down to the single particle level.

"Our work is proof of concept at the moment," Lévy says. "This is an emerging field: we need to develop tools to understand what happens to nanoparticles when they enter the cell." Working with Dr Sée, Lévy has shown that protein-coated nanoparticles are taken into a region called the endosome, where the coating is degraded by an enzyme called cathepsin L. They are now looking at ways to overcome cathepsin L, and also how to get nanoparticles out of the endosome so that they are able to move freely around the cell. Solving these problems will further our understanding of how nanoparticles interact with biological systems in general. This will help inform rational discussion on the safety of nanoparticles for potential applications in nanomedicine, such as drug delivery.

Visualising functional dynamics

Since the 1950s, X-ray diffraction and crystallography have revolutionised our understanding of the molecular structures of proteins. And yet there has been very little progress in obtaining information on the time-dependent conformational changes that are the key to understanding the function of such molecules.

Now, using a new technology called Reflection anisotropy spectroscopy (RAS), physicist Professor Peter Weightman is leading research to measure conformational changes within a protein in real time as it ‘does its job’.

"For 50 years we’ve been characterising the structure of molecules, now we need to look at their dynamics," says Weightman.

Working with BBSRC Professorial Fellow Nigel Scrutton, at the University of Manchester, Weightman has been studying the dynamics of flavoproteins that act as electron carriers between a number of donor and acceptor proteins.

"Previous crystallography studies have shown that flavoproteins consist of two subunits that are joined by a hinge," Weightman explains. "We have shown that, if you pass a charge through the molecule, the hinge unlocks as an electron passes through it and one subunit moves with respect to the other.

"What surprised us was that these motions were much slower than we had thought – in the order of seconds rather than milliseconds."

Weightman believes that understanding both the timing and dynamics of motion of electron transfer could lead to a better understanding of metabolic processes, and could even lead to the design of artificial photosynthetic mimics, which could one day have an application in green energy production.

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