Every living cell is packed with minute molecular machines. These machines copy DNA, produce new proteins, transport them to different locations and generally make a cell work as it should. But how do they do this? This is what Antoine van Oijen, a single-molecule biophysicist at the University of Groningen, is trying to find out together with colleagues from the universities of Delft, Leiden and Amsterdam. They have just received EUR 2.4 million for their work from FOM, the Foundation for Fundamental Research on Matter.
‘The classic way to study the machinery of the cell is in an idealized environment,’ explains Van Oijen. ‘Traditionally, biochemists will look at enzymes in diluted salt solutions, but that’s a far cry from the conditions inside a cell.’ Because cells are literally packed with proteins, DNA and other molecules, both large and small. ‘And that will affect their function.’
Van Oijen, a physicist by training, has been studying the machinery of living cells for the past ten years or so, first at Harvard University and then, for about three years now, at Groningen. ‘Of course, I didn’t know much about biology when I started, but I’ve learned a lot since.’ His office bookshelves filled with biology and biochemistry textbooks show how hard he’s been working on it. But most importantly, he is bringing his unique perspective to the study of living organisms.
Van Oijen has been studying biological processes at the single-molecule level in two ways: in artificial systems and in living cells. ‘We can label a single molecule with a fluorescent dye. This dye will emit light when it is excited by a laser. With a special microscope we can detect this light so we know where the labelled molecule is.’
For example, Van Oijen has fixed a strand of DNA on a glass slide. Using fluid flow, this strand is stretched out. ‘Then, we add a labelled protein complex, which replicates DNA. We can then observe under our microscope how this complex moves along the DNA strand.’
In other experiments, labelled molecules are put back into living cells. Again, they can be localized under the microscope. And by labelling different proteins with different colours, Van Oijen can observe them all at once. ‘So you can see if they co-localize in some particular part of the cell.’
These experiments provide a lot of new information. ‘But there is a large gap between the artificial systems and the living cells.’ Artificial systems provide maximum information on the interaction between single molecules, but – you’ve guessed it – only under artificial conditions. And in the real living cell, you can’t get detailed information on the interaction of the labelled molecules. ‘So we’re going to try and fill this gap.’
One way to do this is to add ‘crowding’ to the idealized systems. ‘We can add high molecular weight substances like polyethylene glycol to these systems to mimic the situation in real cells.’ This will slow down diffusion rates, for example. Adding different protein complexes to a stretched piece of DNA can also provide extra information. ‘In cells, different complexes may be active at the same time and they may, literally, bump into each other. We don’t really know how this affects their function.’
The project has been broken into different parts, which will be taken on by the eight different research groups in the programme. ‘There are actually quite a few single-molecule groups in this country, the fruits of a “physics and biology” programme that FOM started in the 1990s. In fact, our research consortium combines some of the leading groups in the world in the physics of DNA replication.’
The project will employ eleven PhD students, two in Van Oijen’s lab. ‘All the equipment is already there: we just needed the people to do the work.’ He shows us his group’s ten microscopes, all tucked away in special rooms. ‘You can only enter with a keycard, as laser light is involved.’ The laser light is sent to the microscope through a series of lenses and mirrors. ‘And the microscope is fitted with an extremely sensitive CCD camera, which can detect single photons.’
The work that is to be done in the next five years will give us a more realistic idea of the function of biological molecules. ‘With these microscopes we now have the technical ability to do the crucial experiments. But the project will also involve theoretical scientists, who will come up with models to explain our experimental results and tell us what is going on inside a cell.’
The time is ripe to move from single-molecule physics to the physics of the interaction of molecules under realistic conditions. ‘Beyond doubt, the action of enzymes will be very different in realistic environments from in the buffered salt solutions in which biochemists have been working so far.’
And one more thing: ‘Our view of cellular machines is often very static.’ As if a cell were a factory, with all the equipment neatly lined up along orderly transport routes. ‘In fact, these machines are very dynamic. Parts keep being interchanged, as if your car tyres were to be replaced while you were driving it. All proteins and other molecules in a cell are constantly interacting. We hope to produce a more dynamic image of the complexity inside a cell.’
The eight group leaders of this project are:
Antoine van Oijen (RUG),
Nynke Dekker (TUD ),
John van Noort (UL),
Tom Shimizu (AMOLF),
Christophe Danelon (TUD) and
Martin Depken (TUD)
Prof. Slotboom co-applicant in awarded ZonMw project application
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