Today I give a public lecture, curiously entitled, 'Botox, jellyfish and a galaxy of molecules', which is meant to give a flavour of the work that goes on in my research group. I thought it'd be a good idea to try to do the same here - it's turned into a of a whopping blog post but stick with it, I hope you'll find it interesting.

Cell biology, the study of cells and how they work, has undergone a bit of a revolution (if you can have a bit of one of those) in recent years. This has come about because of a better-late-than-never close integration between the life sciences with the physical sciences. This has led to an acceleration in our abilities to actually 'see' what is going on inside living cells (defined as the minimal units of life). Now we can determine the precise locations of single molecules (the minimal units of cells) with nano-scale certainty (there are a BILLION nanometres in one metre), inside living samples.

The gallery below tries to familiarise you with the tiny scales we are talking about - the flea on the pinhead is about half a millimetre long, the orange pollen grains are about 0.1 mm. We can relate to that. The red things are erythrocytes - red blood cells. These are about 8 microns diameter (there are 1000 microns in a millimetre...) - and the green nasties are single E. Coli cells, smaller still at 1 micron long (ie one one thousandths of a millimetre). Living things run out of steam at scales smaller than this - the colourful thing is a viral particle - at around 50 nanometres it is too small to have all the qualifications for being 'alive' but it's made of the same things as cells are; proteins and DNA. Clever biochemistry can determine the position of every atom in a protein (single proteins are between 1- 5 nanometres diameter, incidentally) - but this approach can't be applied to living samples.

The challenge is to determine the postions, interactions and dynamic movements of single protein molecules inside living samples, in 'real-time' - this can only lead to further understanding of cells, diseases and physiology, but needs biology, physics, chemistry and mathematics to come together to make it work.

This is made possible by using fluorescence - the property of some things to absorb light of a particular wavelength (colour) and emit a colour of a longer wavelength. Fancy microscopes can detect these colours with enormous sensitivity, down to single photons. This got really exciting when zoologists discovered that the Pacific jellyfish, Aequorea victoria (named after the great queen, natch) had cells in its body that were fluorescent. Work in the 1980s determined that these amazing light-emitting cells were able to do their tricks using only a single protein - imaginatively dubbed 'Green Fluorescent Protein', or GFP, at that time.

The key point here is that the sequences and subsequent structures of proteins are determined by the sequence of DNA in the cells - so taking the DNA encoding GFP from the jellyfish and putting it into any cell made the recipient green, so making it visible down the microscope. The genetic code being completely conserved across all species means that we can take the gene encoding GFP, fuse the DNA to the gene making any other protein (say, for example, insulin), and the subsequent novel 'fusion protein' made by the cells becomes fluorescent. Now, not only can we see cells, but we can see where groups of individual proteins go inside cells, because we can introduce DNA that we made from a jellyfish gene fused to the gene of something we want to study and get the cells to make a fluorescent protein out of it, that behaves in the way we want it to. Amazing, isn't it?

It gets better. Molecular biologists (the new and better word for 'genetic engineers') and biochemists looked at the structure of the GFP protein and predicted which bits of the protein gave it particular features, like its colour. They then changed the DNA sequence to alter the encoded protein and created a palette of fluorescent proteins that have only ever existed because we made them - and these allow us to specifically illuminate different parts of cells all at once. We've got available to us hues from blue to cyan, green, yellow, orange and reds to play with now, all encoded from different DNA sequences encoding distinct proteins. Beautiful, isn't it?

This has enabled all sorts of things to be seen in living cells, in three-dimensions, over time - things we never saw happening before. An entire software industry, driven by advances in gaming, has sprung up to deal with the very large 3-D computer data files describing microscope images, allowing cell biologists to interact with cells in ways we never imagined even 10 years ago. 3-D glasses were never put to a more productive use.

Things got even better when the physicists wised-up to what we were doing. The fluorescent light we see coming from our new cell-made tools arises from molecules of GFP (or a coloured cousin) - but we can't resolve the single molecules because there are too many of them, too close together, for the microscope to see.Imagine if we could turn off and on single molecules at will, note their positions repetitively until we had resolved the precise location of every molecule in the cell. This way, the light from each individual protein wouldn't overlap so we would be able to determine where it has come from, given sufficient understanding of how the optics in a microscope work.

This idea was made possible using a new form of GFP - a 'photoactivatable' GFP, unable to be fluorescent unless told to do so by the viewer. Shining a controlled amount of UV light onto these proteins randomly 'activates' a subset of the molecules, making them fluorescent - their limited lifetime means they burn bright but die young, until replaced by another subset. Activating proteins, imaging them until they are gone, then repreatedly activating another cohort and imaging those until all the molecules in the cell are consumed, allows us to determine where each light signal arose from. The flashing spots in the movie file below all arose from single molecules like some tiny firework display.

Software mathematically determines the precise centre of each and every one of those spots and so delivers the XY-coordinates of the molecule the light signal arose from - then those coordinates are used to generate a 'map' showing us the location of each single protein. The resulting galaxy of molecules exists in every cell in the body on a scale impossible to imagine. 

Not only that, but of course things move around if the cells are alive. We recently pushed this further, teaming up with physicists and mathematicians who wrote software algorithms to track the fast-moving signals from each molecule zooming around inside cells. You can see this below - the images are inverted because it's easier to see with your eyes, so the dark spots arise from single proteins moving with some purpose or other in the membrane of a living cell. 

Determining the nano-scale tracks that each molecule follows takes some computing power and a lot of time - but it is tremendously informative. None of us could have imagined that we'd be able to measure the speeds and directions 10000s or 100000s of molecules possess inside cells, never mind that it's possible because of a jellyfish.

Why is all this important? What's it got to do with the botox in the title?

Well, protein molecules are the machines of the cell - they catalyse reactions, act as factories to make other components of the cells, form scaffolding and tracks for things to move along as well as countless other tasks. The ones we are particularly interested in are those that regulate secretion - this process is central to all inter-cellular communication and if something goes wrong with it it leads to all manner of problems (such as diabetes and neurological disorders). Biochemists over the decades have defined literally every protein involved in secretion, driven by the fact that it's so important. The final definition of which proteins were absolutely essential came in the late 1980s with the discovery that neurotoxins from Clostridium botulinum bacteria had eveolved to attack specifically sequences only in 3 proteins in the body: those involved with driving secretion in nerve cells. These botulinum neurotoxins are the most poisonous things known on the planet and at the time were used in the lab to prove that the proteins we thought were important were just that, by cutting them with the toxins and determining that their function in cells had gone. With the toxin-cut proteins, the nerves can't communicate with their targets properly, muscles don't work, and you die.

Now we use the toxins for making foreheads smooth. Who could have predicted that?