Guest Post - Don't judge a cell by it's coat

Today's post features a Guest Post from Lucas Brouwers of Thoughtomics. He's written some wonderful science posts (including some great ones about evolution), so I was quite excited when he agreed to write a post for me. Give his blog a look, it's well worth it :)

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As a reader of this blog, you have become closely familiar with a wide variety of aspects of the bacterial world. In this guest-post, I would like to take the opportunity to entertain you with a story on the evolution of eukaryotes (animals and plants), with a big prokaryotic twist!

One of the first things you'll notice when you compare an eukaryotic cell with a bacterial cell, is that eukaryotic cells look incredibly messy. There are all sorts of subcompartments and membranes laying on top of and besides each other. Bacteria look like slick and minimalist living machines in comparison!

Eukaryotes shuttle a lot of proteins and other compounds between these compartments. But since all these comportments are isolated from eachother via membranes, this shuttling requires some special tricks. All eukaryotic membranes contain 'membrane coat' proteins that can fuse together to create vesicles that bud from the mother membrane, and fuse with a target membrane. Inside such vesicle, proteins and compounds can be transported to their new destination. As a picture is worth a 1000 words, and a video even more, you can see the process of vesicle formation for yourselves in the video below.



There are three classes of these membrane coat proteins in eukaryotes, which all share the same three-dimensional structure. A structural biologist would tell you that the features they have in common are a 'beta propeller' and a 'SPAH domain'. Because they all look so similar, scientists believe that these proteins have a common origin. Since they're present in every single eukaryote that we know of, it's reasonable to assume that such a membrane coat protein was already present in the latest common ancestor of all eukaryotes. This last common ancestor would still be very prokaryote-like, so we could expect to find at least some similar membrane coat proteins in prokaryotes. The problem is that up till now, nobody was able to detect these membrane coat proteins in prokaryotes!

The usual way scientists search for similar proteins across species is via sequence similarity: if two proteins in different species roughly have the same amino acids, it's likely that they do the same things in both species. But as time progresses, the differences between such proteins of different species can build up beyond recognition. In this particular case this seems to be the problem, since we're looking for proteins that diverged billions of years ago, before the eukaryotic/prokaryotic split! That is why Rachel Santarella-Mellwig and colleagues decided to take a different approach. Instead of searching for proteins with a similar sequence, they searched for proteins with a similar three-dimensional structure, using structure prediction algorithms.

Surprisingly, they managed to find membrane-coat-like proteins in several bacterial species. All the bacteria where they identified these proteins, belong to the superphylum of Planctomycetes, Verrucomicrobia and Chlamydiae (PVC). Many of these bacteria have the ability to turn their internal membrane inwards, surrounding the DNA with a double membrane (that certainly sounds familiar!). To investage whether the predicted membrane coat proteins also associate with membranes in these bacteria, the team did some further experiments. They choose one of the PVC bacteria analyze in greater detail: the lucky candidate was the freshwater bacterium G. obscuriglobus. The proteins were targeted with gold-coated antibodies, so that they could be visualized with electron microscopy. They found that 95% of the protein localized to the space between the inner and outer membrane, of which more than one third were in close contact with a membrane of a vesicle. If you look carefully in figure five from the paper, you can see small black dots that are the gold particles binding to the proteins (for the people who don't look carefully: there are big black arrows pointing at them). They're located at the edges (membranes) of these dark blobs, which are the vesicles within G. obscuriglobus.
So although the sequences of these proteins have changed beyond recognition, the three-dimensional structure and the overall function of these proteins seem to have been retained through evolution! The authors did not find evidence for an alternative scenario, in which the PVC-ancestor 'gobbled up' the gene from an eukaryote. Thus the likeliest scenario seems to be that a simple membrane folding mechanism evolved in the common ancestor of PVC's and the protoeukaryote, spurred by or accompanied with the evolution of these membrane coat proteins. The authors are a bit more careful in their conclusion, at the end of their paper they write: "... this suggest that the PVC bacterial superphylum contributed significantly to eukaryogenesis". All in all, I think this is a great example of bioinformatics and experimental work coming together in a fascinating story about eukaryotic origins. It makes me wonder what else lies hidden in all databases, waiting for the right questions to be asked...

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Santarella-Mellwig R, Franke J, Jaedicke A, Gorjanacz M, Bauer U, Budd A, Mattaj IW, & Devos DP (2010). The compartmentalized bacteria of the planctomycetes-verrucomicrobia-chlamydiae superphylum have membrane coat-like proteins. PLoS biology, 8 (1) PMID: 20087413

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Guest Posts

I'll be continuing with my own posts, for those of you that miss the enthusiastic monologues about plants and bacteria :p but I am still accepting guest posts to help keep a regular schedule during what's turning out to be an insanely busy term. So if you've ever felt like writing a lab-rat blog post send me an email!

Two Component Systems

For free-living (especially free-moving) organisms, the ability to sense and respond to the outside environment is crucial for survival. Eukaryotes, such as animals and plants, often have highly complex network systems in place to monitor their surroundings and respond effectively, but bacteria have developed a remarkably simple system. It's called the 'Two component system' because it literally relies on just two components; a sensor and a responder. The sensor picks up the signal, communicates this to the responder, which then causes the effect.

The 'communication' of the message from the sensor to the responder, is carried out by transferring phosphate molecules. The signal interacting with the sensor, causes the sensor to autophosphorylate (phosphorylate itself) and then pass the phosphate molecule onto the responder, triggering the response, as shown in the diagram below:


Diagram drawn by me, using all the MS Paint skill I possess. (I've tried to keep the colours colour-blind friendly). Sensor in green, responder in blue, and the brown lines show the path of the phosphate. Blob on the left is the signal molecule that the system is sensing. 'H' and 'D' are amino acids Histadine and Aspartate respectively.

One of the most useful things about this system from a scientific point of view is that the phosphorylated regions are very well conserved across bacterial species. This makes them relatively easy to find, once you have the full genome of the organism, as shown by large-scale searches for two component systems in Bacillis subtilis and Streptomyces coelicolor (both references given below). In both organisms hidden Markov models were used to find the conserved protein sequences, and then sequence alignments carried out to group the sensors and responders into different groups. They also searched for transmembrane domains within the sensors to find whether (and how) they were attached to the surface of the bacterial cell. Unattached soluble sensors suggest a monitoring of the intracellular environment, whereas membrane bound sensors are more likely to provide information about external conditions.

As the function of many of the two component systems (particularly in Strep. coelicolor) is unknown, studies like this provide exciting new avenues of research to explore. One of the main commercial attractions to studying two component systems (ignoring the main attraction, which is simply to find out how the things work) is that they aren't present in animal cells, and therefore could potentially be a target for novel antibiotics. Particularly as many of them are vital for the survival of the bacteria, particularly opportunistic motile pathogens.

In fact, two component systems are very often the way the bacteria senses and responds to the antibiotics as well. Knocking out the vancomycin response system (VanRS) might not kill the bacteria, but combining it with vancomycin treatment would be deadly.

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Guest Posting

Work is sort of getting to be at the moment, now term has properly started there seems to be an awful lot of it happening, and everything is going slightly crazy. So if anyone would like to write guest posts (both blog-owners and non-blog-owners) they would be happily recieved. Leave some form of contact in the comment box (or write to me for those who know my email address) and I'll get back to you.

All I ask is that the posts be vaguelly about science. :)

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Follow me on Twitter!

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Fabret C, Feher VA, & Hoch JA (1999). Two-component signal transduction in Bacillus subtilis: how one organism sees its world. Journal of bacteriology, 181 (7), 1975-83 PMID: 10094672

Hutchings MI, Hoskisson PA, Chandra G, & Buttner MJ (2004). Sensing and responding to diverse extracellular signals? Analysis of the sensor kinases and response regulators of Streptomyces coelicolor A3(2). Microbiology (Reading, England), 150 (Pt 9), 2795-806 PMID: 15347739

Paleoatmosphere

The first signs of life on earth appeared about 4.5 Ga (1 Ga is an American billion, ie. 10⁹ years) ago. It's not yet completely certain exactly how this life arose; hot volcanic mineral springs have been suggested, as have the more traditional lightning-struck primordial soups and (rather wonderfully) radioactive beaches. At any rate something happened (and there was certainly plenty of time for it to happen in) which lead to a little membrane-bound ball with internal nucleic acids which, crucially, could replicate...

Awesome little gif from here

And then it was all over really, bar the evolution.

What kind of world did these first little blobs of life appear in? The surrounding temperature is pretty much unknown, hypothesises for both warm and cold have been put forward and all that can really be agreed on is somewhere between 4-100 degrees. It was most definitely wet; water was in liquid form when life first started, a fact that was probably vitally important for the formation of life as we know it.

The atmosphere would have been very different, little oxygen and an abundance of carbon dioxide with plenty of methane being released into the atmosphere once the first life forms (appropriately called methanogens) started eking out an entropy-defying existence. In order to get energy to power cellular processes you need to set up redox pathways, which involve cycles of electron donors and acceptors. The main electron donors around at the time were H2, H2S and CH4 and the main acceptor probably nitrogenous. Water, the electron donor used for photosynthesis, was around in abundance, but none of the little proto-life-blobs quite had the energy required to split it (or the physical proteins required back then either) so it mostly stayed unused.

Carbon dioxide levels went down, methane levels went up, the planet warmed up a little due to global warming. Things stayed like that for a billion years or so (1 Ga) and then something quite special happened, something that would have mindblowingly devastating affects on the life surrounding it.

Photosynthesis. The uptake of carbon dioxide into the cell, and the reactions that stick it onto a carbon chain, effectively 'fixing' it as sugar; turning air into food. And as everyone hopefully was taught back in primary school, this process releases oxygen, which is good for us but was almost fatal for the life-forms around 3Ga (probably was fatal for some of them). When oxygen isn't being used for respiration, it can be highly toxic to cells. It screws up the internal redox potential, it creates dangerous free-radicals and it precipitates ions out into soluble forms.

Of course the oxygen produced by the photosynthesising proto-bacteria didn't go straight up into the atmosphere right away. There were too many ions floating around in solution to bind to it, and this caused a huge precipitation event; in common terms, everything rusted. Iron was pulled out to form large rust beds, which set down iron ore deposits to be dug up by humanity 2.5 billion years later and used in the Industrial revolution.

The arrival of this new resource (oxygen) lead to a change in the way organisms respired as well. Up until what is sometimes called the Great Oxidation Event (when oxygen started being released into the atmosphere by all the photosynthesising blobs) most respiration was anoxic, probably similar to anaerobic respiration (or fermentation) in anaerobic bacteria around today. This process, while enough t0 keep life going, is around sixteen times less efficient than aerobic respiration. The proto-life-blobs that managed to use the oxygen would therefore have gained a major energy boost.

Over the next 1.5 billion years the atmosphere changed from a highly reducing state (where the early proto-life-blobs developed) to a more oxidising environment. Endosymbiosis and the formation of mitochondria and chloroplasts allowed the first eukaryotes to specialise their metabolism even more. Rather than have the whole cell as a bundle of metabolic redox reactions, releasing potentially dangerous radicals into the cytoplasm, the energy production could be specialised inside it's own compartment, churning out enough energy for the cell to get bigger. Complex intracellular tubules allowed nutrients to be diffused all over this larger cell which would then commit what was from a bacterial point of view the biggest evolutionary mistake ever, and package the cell nucleus away in an inaccessible membrane. (Eukaryote cells then had to develop squishy things like sex in order to regain enough genetic plasticity to actually evolve.)

The effect of oxygen was not just limited to respiration; nowadays many metabolic pathways involve oxygen at some point, including those necessary for the production of sterols (used in signalling molecules and cholesterol, which is an important membrane component), indoles (found in the amino acid tryptophan) and several antibiotics. Oxygen can be an important resource if used correctly.

It's occasionally speculated just why life took so long to move out of the blob phase and into multicellularity. Spending over three billion years as blobs seems a little odd considering that the last billion years involved the branching out of multicellular organisms in a a whole myriad of forms and features, from velociraptors to cockroaches to annelid worms to highly specialised bacteria capable of forming complex networks of bacterial hunting packs. My personal opinion is that all that time was needed simply to get the metabolic background necessary for more complex cellular arrangements. Without the biochemical pathways necessary to generate reasonable amounts of energy, cells have severe limitations placed on their abilities. And biochemically, most organisms are remarkably similar. Differences between the eukaryotes, bacteria and archaea maybe, and plants and fungi have a few different bits of metabolic pathways, but otherwise the internal cellular reactions are remarkably conserved. Not just metabolic ones either; the finely tuned DNA replication machinary, protein synthesis, and even several signalling pathways remain conserved throughout the Kingdoms.

All those internal pathways had three billion years of self organisation and optimisation before they even had to begin to think about making multicellular creatures. No wonder they all fit together so well!

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Falkowski PG (2006). Evolution. Tracing oxygen's imprint on earth's metabolic evolution. Science (New York, N.Y.), 311 (5768), 1724-5 PMID: 16556831

Experimental Lab-ratting

It's starting to approach the time of year when PhD student's need results. Especially those who work on big human-based projects which need human subjects to fill in questionnaires and act as data points. And in the Economics and Law faculties, they are willing to spend a decent part of their budget on funding such studies, given they don't have to buy much else.

It's also the time of year when I start running out of money...

So today saw me heading over to the Law faculty to carry out an experiment in decision making. Not to bore you with the details but it involved being put in an anonymous pair (i.e I didn't know who I was paired with) and being labelled either A or B. B then answered twelve IQ questions and A was given £1 for each correct question. Here's the catch, before the questions were answered, A had to decide how much to pay B for each correct answer given.

Some A's opted for very generous amounts. One person (who either has very rich parents, a very kind heart, or was determined to screw with the experiment statistics) even gave their B the whole sum of £1 for each correct answer.

I was a B.

Apparently there are some people in this world who think that 'fair' means giving someone THIRTY PENCE PER CORRECT QUESTION while walking away with sixty pence yourself for doing bugger all.

I was very, very tempted to deliberately get the answers wrong when I saw that '30' scribbled down on my sheet of paper. But I've been trained to do exams so many times I don't think I ever could purposefully answer questions wrong and, well, I kind of need the money. I mean that was why I was there. Principles of fair behaviour are all very well, but £8.50 (£3 for participating, £3.50 for my questions and £2 for guessing the number of answers I got correct) is £8.50. It would have been hard walking away with just a fiver, although kind of funny to know that whoever-it-was was only getting three pounds for being a tight-fisted... yeah.

Anonymous partner would have ended up with around £12 and can probably work out from this that I got all my answers correct. I hope they're feeling just a little guilty. Or at the very least I hope they have six kids and a mortgage or something.

Huh.

Anyway now I have to go plate out 54 samples. I'm being an experimental lab-rat again on Friday and I've been promised a tenner for that one so yay, I can eat this week :D

Exploring protein interactions: yeast two hybrid systems

>Proteins are one of the key molecules inside cells; involved in signalling, intracellular transport, metabolism and gene control. They rarely work alone, most of the proteins in the cell are part of large complex networks consisting of many interacting proteins. Various techniques exist in order to find these interactions, and one of the most common is the use of yeast two hybrid systems.

Yeast is apparently quite a nice organism to work with (I've never worked with it myself, I must say, apart from a few practicals in second year, where I almost set my lab-partners hand on fire). The genome is fully annotated, the organism is well characterised, and yeast grows and responds quite fast, so experiments shouldn't take too long.

The yeast-two hybrid system is based around molecules called transcription factors, which are normally used by the cell to active gene expression. In yeast, there are several transcription factors which consist of two separate molecules, which need to be in close proximity in order for a gene to be expressed. If you attach an experimental protein to one half of the transcription factor, and another experimental protein (that you think interacts with the first) to the other half you can test for interactions. If the two proteins do interact then the transcription factors will be brought close together and the gene downstream of them (which acts as a reporter gene) will be expressed:The diagram above (taken from the reference below) shows this process. The blue jigsaw-shaped 'X' and 'Y' proteins are the experimental proteins, being tested for interactions and the yellow shapes are the two parts of the transcription factor. The big white cloud is the polymerase, which begins the process of turning the DNA into protein. The reporter gene can be set to code for a vital compound such as histidine; stick the whole system into a histidine deficient mutant and you have a marker system to see if the proteins interact. If they do, the histidine is produced and the cells can grow, if they don't, the cells die.

One of the most useful things about this technique is that it can be automated, and used to scan whole libraries of proteins to see if they interact. By using a matrix, each protein X (the 'bait' protein) can be given a defined position and then systematically exposed to a number of different protein Y (the 'prey' protein). If you're taking Y from a clonal library, and have a sufficiently intelligent robot, the whole procedure can be carried out with minimal human input.

The yeast two-hybrid system has been invaluable for determining many important protein-protein interactions however there are some problems with it. Firstly, this is a yeast two component system, and most protein complexes consist of many interacting proteins, certainly more than two! Secondly, this whole system relies on two soluble proteins interacting in the nucleus (where the DNA is) and so doesn't work for membrane bound protein interactions.

In view of this, several modifications have been made to the original methodology to make it more useful for trapping a wider range of protein interactions. It's been expanded into the three component system, which identifies proteins that interact with (or inhibit) both the the bait and the prey. Using another natural yeast system (the G protein system) has allowed transmembrane proteins to be identified as well:
In this system protein Y contains a binding site for a subunit of the G protein, while X is a membrane-spanning protein. G proteins are membrane bound proteins that activate transcription factors inside the nucleus. If the two proteins interact then the G protein subunit bound to the Y is sequestered away from the rest of the complex, and the G protein signal cannot be transmitted. There is therefore no signal to the transcription factors, meaning reporter genes (in this case genes for pigmentation rather than for death) will be turned off.

Even with these modifications there still are problems with the yeast two hybrid system. There is very little quantitative analysis involved to see how strongly the protein is binding, and the discovery of new interactions will always be limited by the choice of proteins to screen. However one of the largest advantages of yeast two hybrid systems is that it carries out protein binding analysis inside the cell, in proper cellular conditions, unlike affinity binding which, while it can identify much larger protein complexes, involves taking proteins out of the cell and handling them in vitro.

Yeast two hybrid systems are therefore still one of the main practical methods used for determining protein interactions. The field of systems biology is a fascinating one, and while attempting to catalogue the whole spectrum of cellular interactions may seem like a daunting task ('interactomics' for the funding people) working towards it will only reveal more and more useful information about the complex and fascinating networks of proteins within the cell.

[btw: The reference below is a great source of information about the many different modifications made to the basic yeast two hybrid system, thoroughly recommended for anyone interested in protein interactions or systems biology]

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Brückner A, Polge C, Lentze N, Auerbach D, & Schlattner U (2009). Yeast two-hybrid, a powerful tool for systems biology. International journal of molecular sciences, 10 (6), 2763-88 PMID: 19582228

Half plant, half predator, all weird.

I was planning on brushing up my knowledge of chloroplasts today, as next week I'm starting a plantsci course for my options lectures, but I got sidetracked by Captain Skellet alerting me to Hatena. I've heard of several organisms containing proto-plasmids; symbiotic chloroplasts which haven't completely been endosymbiosed, but Hatena was a new one so I went to look it up. And I'm very glad I did, because it's pretty amazing.

Hatena, taken from the reference. Green blob is the symbiont. Scale bar is 10um

Quick background: chloroplasts are little membrane enclosed vesicles in plants which carry out photosynthesis. Current theory for how they developed is that they were once free-living bacterial type organisms (cyanobacteria) which were engulfed by a larger cell and over time lost their own identity and became little photosynthesising factories inside the larger cell. (I've got another post on it here for anyone particularly interested in the subject.)

Hatena arenicola doesn't have a chloroplast, but it does have a symbiotic relationship with another organism; nephroselmis. The nephroselmis is always found in the same place in the Hatena, and carries out photosynthesis to provide energy for both of them. Unlike regular chloroplasts, nephroselmis has it's own proper nucleus and even it's own mitochondria although most of the internal cellular organisation and any kind of motile apparatus (such as flagella) has been lost.

The weirdest thing about these two organisms though, is their replication cycles. When Hatena replicates, the nephroselmis doesn't, and as a result only one of the offspring gets the photosynthesising symbiont. The other organisms remains colourless and develops a complex feeding apparatus at the apex of the cell, presumably as it can no longer rely on the symbiont for food. This wierd 'half plant, half predator' lifecycle is shown below. (Picture taken from the reference, scale bar 10um):


That's just weird. Seriously odd. The Hatena is able to move seemingly freely between being a predator consuming other cells for food, and being a plant-like organism, once it settles down with it's symbiotic partner. The grey non-symbiont organisms can be induced to take up free-moving nephroselmis and (in the words of the paper) "tentitavely" maintain a symbiotic relationship with them.

The paper suggests that Hatena cycles between these two modes of living, depending on circumstance. Thus the 'predator' grey cell shown above will continue eating fellow cells until it consumes a nephroselmis, at which point it degrades its complex feeding apparatus, accepts energy from the symbiont until it's ready to divide. One of the daughter cells will then go through the whole cycle again while the other remains as a non-predating plant. The authors freely admit that there is little evidence for much of these stages, but it seems a reasonable way to explain what is going on.

As this is clearly a very early stage in symbiotic capture it has important implications for the endosymbiotic theory of chloroplast evolution. Along with various other 'intermediate' symbionts (such as Karenia mikimotoi and Lepidodinium viride) the Hatena helps to show how chloroplasts might have first formed in the cellular ancestor of plants. Hatena and its symbiont have already acquired an intimate structural association, only the coordination of their cell cycles would be required to turn the nephroselmis into an internally replicating plastid.

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OKAMOTO, N., & INOUYE, I. (2006). Hatena arenicola gen. et sp. nov., a Katablepharid Undergoing Probable Plastid Acquisition Protist, 157 (4), 401-419 DOI: 10.1016/j.protis.2006.05.011

Cycling Cells - The circadian rhythm

Circadian rhythm is the cyclic control of cellular processes over a period of roughly twenty-four hours. There are many processes within the body that are held under circadian control; the need to eat and sleep, blood pressure and some hormone production to name a few. Circadian control is an important development in evolution, as it allows behaviour to adapt to appropriate times in the day. Humans have not adapted to function particularly well at night, so using that time for sleeping means they can be more alert during the day and in the hour or so before you wake up (on weekdays at least), the body gets busy increasing the blood-pressure, preparing you to need the bathroom and, slightly bizarrely, increasing testosterone levels.

I wasn't planning on doing a post on circadian rhythms, but Alejandro mentioned it in one of the comments, so it was in the back of my head when I was looking for a paper to review this week. And it's about time I started getting back into more general molecular biochemistry (less than five months left till exams!) rather than concentrating exclusively on bacteria.

From a cellular point of view circadian rhythms are controlled by careful feedback loops between interacting proteins. In this paper, the main positively regulating proteins they were looking at were proteins named CLOCK and BMAL1, which bind to promoter sequences of circadian rhythm genes and switch them on. They also turn on the negative regulators PER and CRY which, when they get to high enough levels, bind to CLOCK and BMAL1 and stop them from functioning, turning the circadian rhythm genes off again.

Many clock proteins undergo post-translational modifications in order to give a further level of control. In particular they can be phosphorylated (a phosphate group is added onto the protein) by proteins known as protein kinases. The main work of the paper I decided to look at (reference below) was identifying GSK3β, a protein kinase which phosphorylates BMAL1. Like most kinases, GSK3β is not just involved in circadian rhythm control, but takes part in many other cellular functions such as control of glucose homeostasis, cell fate determination, and cell survival. It's not surprising, therefore, that is can be involved in a number of pathological conditions, including diabetes, Alzheimer's disease, cancer and bipolar disorder.

The first study done for the paper was to show that GSK3β will bind to BMAL1 and phosphorylate it. This was done by adding pure samples of the proteins together, along with radiolabelled ATP as a source of phosphate. The result is shown below (all diagrams taken from the reference at the bottom) wiith GST used as a control to check what the GSK3β does when there's nothing for it to phosphorylate (it phosphorylates itself):


As only radiolabelled material shows up on the autoradiogram, this shows that the GSK3β has transferred the radiolabelled phosphate onto the BMAL1.

For further proof that the GSK3β was carrying out the phosphorylation under physiologically relevant conditions, cell lines were used which contained no GSK3β (-/- mutants). Comparing the levels of BMAL1 in these cells with wild type cells showed higher levels of BMAL1 in the mutant strain. This is expected as the addition of the phosphate is thought to lead to the addition of another group, ubiquitin. Ubiquitin is (as far as I am aware) an almost universal signal for 'Degrade This Protein'.

The next stage was to look for the actual sites of phosphorylation; the places on the BMAL1 where the GSK3β sticks the additional phosphate group. As with most kinases, GSK3β recognises a specific pattern of protein residues (T/SXXXS/T for anyone who's interested) and fifteen of these sequences were found in BMAL1. The exact residues were found by point mutation; certain amino-acids were changed and the resulting change in phosphorylation measured:
The wild type protein is shown on the left and the mutant, with the significant T residue converted to an alanine(A) on the left (the change is notated as T21A; the T in position 21 has been changed to an A). This change in one amino acid has decreased the phosphorylation by 40%, although it would be nice to see some actual values rather than relative ones.

The final test to confirm that GSK3β is involved in circadian rhythm control of BMAL1 was to show some actual cyclic behaviour of the protein. For this two cell lines were used, the wild type and the GSK3β -/- mutant. α-tubulin was used as a control; this protein is expressed at constant levels over time and therefore shows that the decreasing and increasing levels of BMAL1 is actually due to changing levels in the cell, rather than just a smaller cell sample containing fewer proteins overall:
That's quite a beautiful gel; in the wild type cells the BMAL1 cycles nicely over the time period (they don't run the experiment for terribly long, but BMAL1 cycling has been proved adequately elsewhere, and the wild type cells are more of a control than the actual experiment). When you knock out the GSK3β, however, the cycling pretty much stops. The paper is careful to point out that is doesn't completely stop, some evidence of differing levels is still seen, but this is to be expected. It's very rare that important cellular processes in mammals are placed entirely under the control of one protein, and there are likely to be other pathways involved in the circadian control of BMAL1.

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Sahar, S., Zocchi, L., Kinoshita, C., Borrelli, E., & Sassone-Corsi, P. (2010). Regulation of BMAL1 Protein Stability and Circadian Function by GSK3β-Mediated Phosphorylation PLoS ONE, 5 (1) DOI: 10.1371/journal.pone.0008561

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For those interested in such things, I now have a twitter. Like the blog, it will be strictly sciency, rather than personal and will be updated far more often.

Wonderful Life - book review

This is my first attempt at a proper scientific book review, so any feedback would be much obliged. Thanks!

The Burgess Shale is a collection of fossils of soft-bodied invertebrates that was formed soon after the Cambrian Explosion, an apparently rapid appearance of a many groups of complex animals. Initially discovered in 1909 by Charles Walcott, the fossils were later examined by three researchers in Cambridge; Charles Whittington and two graduate students, Derek Briggs and Simon Conway Morris. What began as an exercise to catalogue a group of ancient arthropods turned into the discovery of several new phyla of organisms, and ultimately changed the way evolution and selection were viewed. “Wonderful Life” by Stephen Jay Gould is a documentation of this process, exploring and explaining this new view of evolutionary change through Deep Time.

Although Gould was not personally involved in the cataloguing of the Burgess Shale organisms, he was in close communication with the people who were and his clear enthusiasm for the subject shows through. In the preface Gould sets out three main aims; to chronicle the intellectual drama that *was* the Burgess Shale examination, to explore the implications that this change in the perceived workings of evolution brought about, and finally to briefly look at *why* the discovery of the Burgess Shale seems to have passed so unnoticed by the general public, and even non-paleontological scientists.

The book is divided into five sections. The first sets up the central theme that the book is out to destroy; the iconographic idea of the March of Progress, that evolution is a kind of ‘onwards and upwards’ affair, with each generation leading to greater complexity. This was the idea that the discovery of new creatures in the Burgess Shale, creatures that belonged to no known phylum (and could therefore not be simpler and less developed forms of current animals) began to destroy. The second section covers background information considered necessary for an understanding of the Burgess Shale, a quick course on the Paleontological timeline and arthropod anatomy. Being the rather badly specialized microbiologist that I am, I read through these dutifully and promptly forgot them, so can safely say that it’s perfectly possible to enjoy the book without a great depth of scientific understanding.

The third section was for me the most exciting, as it was the actual description of the Burgess Shale creatures, written in order of their discovery (by Wittington, Briggs and Conway Morris). As they slowly discovered more new creatures, they began to realize that these were animals that had never been seen before, that had been wiped out by some extinction event. Furthermore, there was no particular evolutionary *reason* for certain animals to have been saved, the ‘March of Progress’ was beginning to look more like a lottery of chance. The last part of this section discusses the implications of this point of view, that humanity is not a strived-for evolutionary point of perfection, but simply a small twig on a tree of life which has had several branches snapped off altogether at different points in time.

The fourth section leaves the Burgess Shale (in a rather anticlimactic and in my opinion a slightly disappointing shift) to discuss Walcott, the man who found the fossils in the first place. Being a busy man, who in later life was caught up with various family tragedies, Walcott never properly had time to examine his fossils, and in the few papers he did write about them, he ‘shoehorned’ every fossil into modern phyla. Although I couldn't find the discussion of the life of an Edwardian scientist anywhere near as exciting as the beautiful fossil-creatures of the Shale, this section allowed a detailed examination of the shift in the way evolution was viewed as a theory, and why the original iconography of the March of Progress was so seductive and successful. In the fifth section Gould takes a brief but fascinating look at how things might have changed had life had a chance to play out a second time, if different branches of the tree of life had been cut off at different stages in history.

Overall I really enjoyed the book; I was especially pleased as this is (embarrassingly) the first book by Stephen Jay Gould I’ve ever read. The writing style is easily accessible, even to people with a very sketchy view of Deep History and the importance of arthropods, and is, a little surprisingly, highly immersive. The creatures of the Burgess Shale are so beautiful and wonderful that they stand up perfectly well on their own, and Gould lets them do so, his writing concentrating on exploring the philosophies surrounding their discovery rather than over-elaborate descriptions. The many accompanying pictures, most of which are the original drawings made by Wittington taken straight from the fossil samples, help to provide a wonderful visual image of the amazing and sometimes quite frankly weird creatures that populated the Burgess Shale.

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For those interested in such things, I now have a twitter. Like the blog, it will be strictly sciency, rather than personal and will be updated far more often.

Obligatory January The First Post

I like organising myself, which may come as a surprise to people who know me. When it comes to revision time I like making revision plans, when it comes to holidays I like planning what I'm going to do each day, and when it comes to New Year I tend to go a bit crazy with the resolutions.

What I am not very good at is actually doing things. I'm good at starting things, just not sticking with them. The main problem with all my wonderful plans is not so much that they aren't good plans, but that I never follow them through. I'm surprised I'm still blogging to be honest; over one and a half years now when most of my new-exciting-idea-things barely make it to the six month mark.

So this year, instead of making a whole page of resolutions and then breaking them all by February, I'm going to make just six, and really try and stick with them. Three things I won't do, and three things I will.

Things I will not do next year:

1. Waste time on the Internet. I'm not talking about the fifteen minutes morning webcomic-check, or an hour spent browsing on scienceblogs or research-blogging. I'm talking about four hours looking up TV-tropes kind of time wasting.
2. Spend longer than fifteen minutes in the shower. My current average is thirty minutes. My getting-up routine consists mostly of shower. This is not good.
3. Buy non-fairtrade chocolate. This one is so hard not to break. It's amazing how you can be half way through a Twix before you realise you're eating forbidden chocolate.

Things I will do next year:

1. An hours work every evening (at least), unless I am out of the house. Taking an evening to go out with friends (or just the one special friend :D ) is fine. Spending the whole evening lying on the sofa breaking the other resolution-number-one is not.
2. Go to Yoga at least once every two weeks, ideally every week. I tried to do this last term, but things kept getting in the way. This term I should be able to keep it up more regularly.
3. Blog regularly. Ideally two posts a week, dropping back to one post a week in times of Stress (due to exams, job-hunting, etc.)
   

My holiday is officially over now, as is the Hiatus. I have so many research-blogging posts to catch up with it is unreal.