More Fluorescent Proteins

Although I’m approximately two years late in reporting about this discovery I still think it’s pretty cool. In 2013 Kumagai et al. for the first time discovered a fluorescent protein in a vertebrate, the Unagi eel. Until then fluorescent proteins had only been found in invertebrates, such as reef corals and the jellyfish Aequorea victoria, where the famous green fluorescent protein originally came from. My attention was drawn to this finding in a brief article in the Chemistry World journal, in which the author claims that, “Unagi’s status as a culinary delicacy means you’re more likely to encounter these eels in a restaurant than a lab”. [Picture copied directly from the article link.]

Apart from (presumably) tasting good and looking pretty, the fluorescent protein – called UnaG – from Unagi eels may be able to form the basis for a diagnostic test for liver disease. UnaG only fluoresces when bound to bilirubin, which is a break-down product of haem, the molecule that carries oxygen in the blood. Livers with impaired function have difficulty further processing the bilirubin before it is excreted, leading to a build-up of bilirubin in the body, and in extreme cases to jaundice. So the intensity of UnaG fluorescence can be used as a read-out for how badly the liver is damaged.

And now for something completely different: next time I’ll be writing about what it’s like to be an intern at the journal eLife!

Reference:

Kumagai A, Ando R, Miyatake H, Greimel P, Kobayashi T, Hirabayashi Y, Shimogori T, Miyawaki A A Bilirubin-Inducible Fluorescent Protein from Eel Muscle. Cell 153: 1602-1611

Departmental Research Day & M. Sci. Symposium

The beginning of Lent term was far from gentle. For three days I have been sitting in lecture theatres and seminar rooms. Firstly, for a full day we listened to several professors/group leaders of the biochemistry department describing their research. And secondly, we had two days of the so-called Part III symposium, that is a twenty-minute research update from each of the 31 biochemistry M. Sci. students in the department. (Since then I have started working in a lab again and I have to admit I had forgotten how strenuous it can be.)

First things first. The “departmental research day” was hosted at Robinson College, because the lecture theatre within the department is actually too small to seat all the members of staff and students. The introduction was given by Chris Smith and his most interesting point was probably that the department received an Athena SWAN bronze award last year, which “recognises and celebrates good practice in recruiting, retaining and promoting women in Science, Technology, Engineering, Mathematics and Medicine (STEMM) within Higher Education”. So three cheers for the department!

The actual research talks by the various professors ranged from mildly piquing to downright riveting. There were several talks on cancer (the head of the department, Gerard Evan, is a cancer biologist so this is hardly surprising): at one end for example, Helen Mott explained how basic biology, crystallography and peptide chemistry are being exploited to research a new class of drugs based on alpha-helical peptides, which are meant to block activity of some small GTPases (sometimes known as cellular switches because they can turn signalling pathways on and off). At the more clinical end, Kevin Brindle demonstrated how techniques such as dynamic nuclear polarisation magnetic resonance imaging (MRI) are progressing to better image biology/cancer in (live) patients. However, the department is also strong in the field of structural biology, since the crystallographer Tom Blundell used to be the head of the department. Furthermore, there is an increasing number of lab groups working on single-celled eukaryotes such as trypanosomes and Toxoplasma.

Additionally, there were at least two overt political references to keep us on our toes. The first one was this:

And I have to say that I wholeheartedly agree. Perhaps unsurprisingly, the professor who used this image in her slides is originally from the Czech Republic and probably quite vehemently opposes the idea of having an in/out referendum in the UK. [Eukaryotes, by the way, are organisms whose cells contain a nucleus and would include plants, animals and fungi, but also single-celled eukaryotes such as trypanosomes and Toxoplasma.]

The second political reference was a quote by Donald Rumsfeld: “As we know, there are known knowns; there are things we know we know. We also know there are known unknowns; that is to say we know there are some things we do not know. But there are also unknown unknowns – the ones we don’t know we don’t know.” He said this in response to questions about Iraq’s involvement in the supply of weapons to terrorist groups. In the context of science this was a comment on the inherent difficulties of modelling biological processes: Steve Oliver uses yeast as a model organism to study metabolic pathways, and in contrast to the qualitative modelling I have been doing, his models are quantitative (i.e. use differential equations and enzyme kinetic data). Interestingly however, both types of model can suffer from similar problems; for example they can be plain wrong, or incomplete, or based on faulty assumptions. And sometimes when we know they are wrong that doesn’t mean we know how to improve them. Not knowing that they are wrong/incomplete (i.e. the unknown unknown) is arguably the most comfortable position to be in.

The following two days were filled with project reports of all the biochemistry M. Sci. students. It is worth noting that several of these talks were possibly more interesting and of better quality than some of those given by the professors. There was an extremely wide variety of topics including: cancer research, developmental biology, disease biology (including the rare lysosomal storage disease called Krabbe disease), in vitro enzyme evolution, structural biology (including taking trips to the x-ray source near Oxford, the Diamond Light Source), stem cell biology and research into the origins of life. This latter research project is investigating how the first RNA molecules may have come together to form larger, catalytic molecules of RNA (“RNA world hypothesis”), and to do this the reactions are carried out at -9ºC in the eutectic phase of water-ice, a condition thought to mimic prebiotic chemistry.

Lastly, what would a blog post be without the mention of CRISPR. At least two of the M. Sci. projects involve the use of this genome-editing technology. In one case it will be used to knock-out a microRNA that may be involved in the regulation of bicoid mRNA during Drosophila (fruit fly) development. And in the other case it is being used to target a transcription factor that is implicated in the regulation of stem cell fate. Interestingly, the strategy here involves using two guide RNAs simultaneously, both targeted to within the gene of interest, with the aim of creating a large deletion rather than just a small insertion/deletion.

Needless to say, the progress of all our projects is far slower than we (and probably our supervisors) would have hoped.

The Nobel Prize in Chemistry 2014

Since my knowledge and understanding of neurobiology are rudimentary at best, I thought I would write a brief entry on the Nobel Prize in Chemistry instead. This year’s prize was jointly awarded to Eric Betzig, Stefan W. Hell and William E. Moerner “for the development of super-resolved fluorescence microscopy”. [A lot of the information in this entry came from here.]

Principally, one can distinguish between two types of super-resolved fluorescence microscopy: either when an ensemble of fluorophores (those chemical entities emitting light upon appropriate irradiation) is involved or when single fluorophores are being imaged. The former technique came into use in around 2000, while the latter has only been available since 2006; there seems to be a general trend towards shorter timespans between when the scientific discoveries/developments of a technique are made and when the prizes are awarded.

Why bother with super-resolved fluorescence microscopy in the first place? It’s such a mouthful that it might not be worth the effort to develop. However, if you have a look at the following image, entitled “Untangling Neurofilaments” and submitted to the Cell Picture Show by none other than Stefan Hell, then you might realise that it is a really useful technique [image copied from the Cell Picture Show website].

The image on the left shows neuronal filaments and was acquired using confocal fluorescence microscopy, a technique that is already more advanced than standard or epifluorescence microscopy because it only allows visualisation from a single focal plane, thus increasing contrast and resolution. On the right, however, the same filaments are imaged using a super-resolved technique called stimulated emission depletion (STED). Clearly, this second micrograph shows a lot more detail and one can distinguish different filaments from one another. This can be important when trying to distinguish pre- from post-synaptic neurons, for example.

The main problem in optical microscopy, as alluded to above, is that of the diffraction limit: two objects that are closer to one another than approximately half the wavelength with which they are being visualised cannot be distinguished. For example, when imaging with blue light (the light with the shortest wavelength before it becomes UV light) two objects closer than 200 nm (400 nm divided by 2) will appear to be a single object. And this is the theoretical limit; in practice the resolution is worse. [For a more rigorous definition of the diffraction limit and a more physics-based/mathematical discussion I would recommend the “Scientific Background” provided by the Royal Swedish Academy of Sciences.]

A typical bacterial cell is about 2000 nm by 500 nm and so by conventional light/fluorescence microscopy these cells can be visualised, but their internal structure cannot be resolved. Super-resolved fluorescence microscopy relies on visualising only a subset of all fluorophores in a given sample and being able to pinpoint more precisely from where the photons are being emitted (the physical explanation of this, I’m afraid to say, is beyond my capabilities; best to check here and in the references there, if you are interested).

However, something I can explain links perfectly back to one of my favourite classes of proteins, the fluorescent proteins (FPs). When William Moerner was studying green fluorescent protein (GFP) mutants from Roger Tsien’s lab (Nobel Prize in Chemistry 2008), he noticed that some of them had to be activated before they would fluoresce at all and could be irreversibly turned off as well. This allowed the switching on of only a subset of FPs and the detection of these at super-resolution. Subsequently, this first subset is switched off and the next subset turned on, so that sequentially all FPs can be imaged and the whole picture put together. The method is called PALM (Photoactivated Localisation Microscopy).

Just like with the CRISPR Craze, I first heard about these microscopy techniques last year in a supervision: one of the M.Sci. students was presenting her project which involved PALM imaging of an enzyme as it moved along DNA; to control for the fact that the DNA itself might be moving she tracked a histone variant (histones are proteins that associate with DNA to facilitate its tight packing) and used it as a proxy for DNA movements.

Lastly, although this intellectual and technological achievement certainly deserved to be recognised by a Nobel prize (but why the Chemistry prize I’m not quite sure – Medicine/Physiology or Physics actually both seem appropriate too), I wonder how “right” or “fair” it is to award these prizes to individuals. On the one hand, yes, these three men all had great ideas about how to improve light microscopy, but on the other hand, they presumably had a tremendous amount of help from their lab members. Furthermore, science today is far more collaborative than it was when Alfred Nobel lived. I wonder whether there is a way to make sure more people are acknowledged for their hard work…

Oh, and have I mentioned that I love cells? If you do too, then maybe one of the activities at Biology Week will tickle your fancy.

The Green Revolution aka GFP

In 2008 Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry “for the discovery and development of the green fluorescent protein (GFP)”. After its initial discovery in 1955 a lot of luck and hard work were necessary to push GFP to the forefront of a “green revolution” in biotechnology.  “Green” in this context does not refer to environmentally friendly or ecological, but rather to the actual colour: GFP is a protein (a fundamental building block of all living cells) that emits green light after excitation with blue light.

Why am I writing about GFP today? The protein and the revolution it has sparked are no longer really “topical issues” in biology, but GFP is so widely used that everyone knows about it and furthermore, I owe at least some of my interest in the life sciences to this remarkable protein.

Firstly, Osamu Shimomura discovered the protein in a species of jellyfish called Aequorea victoria, which, I feel, somehow bodes well. Secondly, the first time I ever wielded a micropipette or ran DNA on an electrophoresis gel was during a summer science camp at the Vienna Open Lab (http://www.openscience.or.at/#!/vol) in 2008. The one-week project was meant to introduce us rebellious and adolescent 15-year-olds to various molecular biology techniques, including microscopy, DNA restriction digestion and ligation, and polymerase chain reactions. The tutors taught these techniques by showing us how to engineer bacterial cells, Escherichia coli, so that they express GFP and would glow upon UV irradiation. Note that this was shortly before the announcement of the Nobel Prize that year. So here I am loading my first agarose gel (not entirely sure why I wasn’t wearing a left glove…). And the picture below shows the glowing bacterial cell pellet at the end of the week:

In and of itself this experiment was not useful (except that it relieved our burnt-out parents for a week) since fluorescing bacteria on their own don’t do much. However, the experimental techniques that we performed are standard procedures of molecular cloning (the manipulation – cutting/copying/pasting – of DNA, rather than making an exact copy of an organism).

Thirdly, when I entered the last year of secondary school I had the option of undertaking an “independent research project” and I chose to attempt one in Chemistry. The “thesis” – it’s really more like a long review article – was entitled: Green Fluorescent Protein – A Biochemical Perspective. I chose the topic because  of all the fascinating things out there in the science world, GFP was at least something I had already heard about/had some exposure to.

Lastly, since the start of my “real” research experiences I have worked with fluorescent proteins several times. Initially I thought that it was lucky to have landed in a project that utilises GFP, but in hindsight I realise that the fluorescent protein toolbox is so ubiquitous that it is difficult to evade. For example, last summer I was trying to find out where exactly within a fission yeast cell a certain protein was located: to do this I tagged this protein of interest with GFP (in practice this means fusing the gene encoding the protein of interest with the gene encoding GFP), which I could then visualise using a microscope. Wherever I saw a a green dot I assumed that the protein of interest was also present. To pinpoint the exact location of the protein of interest I needed to see a reference protein whose location was known. To this end we chose a protein called Mis6, which is found at a DNA structure called the centromere from where the two copies of DNA are pulled apart during cell division, and fused it to a red variant of GFP called mCherry. Now I could look down the microscope and simultaneously visualise green and red dots, and see where they were in relation to one another.

This summer GFP and I crossed paths yet again. The development of a new technology called CRISPR, which will be discussed in a separate post, is in the process of revolutionising molecular biology again, and its application in combination with fluorescent proteins is/will be extremely useful. In particular, the expression of fluorescent proteins inside cells (for example together with the expression of CRISPR components) allows machines to physically sort fluorescing cells from non-fluorescing cells in a process termed fluorescence-activated cell sorting. This in turn simplifies things for the researcher who can then only work with the fluorescent (i.e. biologically interesting) cells.

The above two examples only give a glimpse of how the fluorescent proteins are used in research today, but one other application that I find aesthetically pleasing is the so-called Brainbow, in which different types of neurons are labelled with different fluorescent proteins to yield images like this (from http://cbs.fas.harvard.edu/science/connectome-project/brainbow#):

The bottom line is that “seeing is believing” and a lot of science seems more convincing when we can actually look at an image of a (live) cell and convince ourselves of our hypotheses (or not, as the case may be). Not to say that there aren’t any caveats with using fluorescent proteins, including, for example, that some cells produce fluorescence of their own accord that may obscure exogenous expression of GFP.

Interestingly, although GFP and its cousins are now used routinely in virtually all molecular/cell biology labs around the world, the original function of the protein is still unknown. If you are interested in reading more about the “green revolution” then I recommend the popular science book “Glowing Genes” by Marc Zimmer or one of the many review articles available online (e.g. Kremers et al. (2011) Journal of Cell Science).