Of moss and me

I recently had a look through some new papers published on moss (Physcomitrella patens) and found one published in Cell titled ‘Transcriptional control of gene expression by microRNAs’ [1]. This didn’t seem to fit the same pattern of a lot of moss papers I have read (ie in less high impact journals). This paper shows how useful moss is for studying gene expression using reverse genetics (so hopefully my PhD wont be a failure).

I think it is important to study important biological problems, such as RNA silencing and epigenetics, in a diverse range of organisms. I take this title from a short review published in Nature Medicine recently (as well as the Steinberg book) by Sir David Baulcombe (who holds a prestigious chair at Cambridge). He wrote about what we have learnt by studying things in plants (eg discovery of transposons) and their importance as a model, using his own research into RNA silencing and epigenetics as the main example [2]. I think this new moss paper goes some way to support this, but also to show the importance of this particular organism. My choice to work with Physcomitrella was because I wanted to stay in the plant world but wanted to work with something where I could knock out specific genes by homologous recombination and was simple to grow. Physcomitrella can be grown on simple agar media plates and joins the few species where genes can be knocked out.

Khraiwesh et al (2010) looked at what happened when you knocked out two Dicer-like (DCL) proteins in moss [1]. Dicer proteins in animals and plants cut long dsRNA to make small RNAs loaded onto Argonaute proteins (producing the effector complex). This effector complex is what causes the target mRNA to be cut or repress its translation. When PpDCL1a was knocked out you see a lack of microRNAs (miRNAs) being produced and the plant is restricted to its first developmental stage only. Not surprising. Strangely, when you knocked out PpDCL1b get production of miRNAs but not cleavage of the targets. It has been shown in animals, but not other plants, Dicer proteins can sometimes be used to help a small RNA in the effector complex to cut the target, as well as in miRNA or siRNA biogenesis. Very interestingly they found these targets had a lower level of expression in the mutant plants. If cleavage is not reducing the mRNA levels then what? The answer to this as many other odd questions in biology is epigenetics. Well DNA methylation at least. siRNAs have been shown to cause gene silencing but not much evidence for miRNAs doing this before. The authors went onto show the ratio of miRNA to mRNA target is critical. If you only have a little miRNA compared to mRNA target you get cleavage. If you have lots of miRNA to target mRNA it causes DNA methylation and silencing. But the methylation was only seen in the knockout so why should we care?

Finally they end the paper by showing ratio of miRNA to target mRNA is important in an endogenous gene involved with a hormone response. The plant hormone ABA mediates drought response and abiotic stress. In moss, when ABA treated you get a decrease in a transcription factor response gene. This is a target of a natural miRNA. This down-regulation is due to an increase in miRNA expression, changing the effect from cleaving the transcription factor’s mRNA to targeting its promoter for methylation and silencing expression. This study shows that miRNAs have the potential to cause DNA methylation, which had not yet been show, as well as suggesting a mechanism for it. Future studies can now go on to show the relevance of this in other systems from yeast to flies to mammals.

I end by simply saying MOSS ROCKS.

Khraiwesh, B., Arif, M., Seumel, G., Ossowski, S., Weigel, D., Reski, R., & Frank, W. (2010). Transcriptional Control of Gene Expression by MicroRNAs Cell, 140 (1), 111-122 DOI: 10.1016/j.cell.2009.12.023

Baulcombe, D. (2008). Of maize and men, or peas and people: case histories to justify plants and other model systems Nature Medicine, 14 (10), 1046-1049 DOI: 10.1038/nm1008-1046

Learning how to tell time with cyanobacteria

Most living things can innately tell the time. If you take a person and put them into continuous darkness for months by themselves they will follow a sleep-wake cycle of about 24-25 hours (as well as going a little mad). Circadian rhythms allow life to predict the raising and setting of the sun to allow us to change our gene expression metabolism and behaviour with the changing of the day. Predicting changes in temperatures and light levels are very important. Circadian clocks have three important features. First of all they are free-running at about 24 hours (in constant conditions like light or darkness they will keep going at ~24 hours). Secondly they are temperature compasatory meaning that changes in temperature do not greatly affect the clock. Finally they must be able to be reset by environmental inputs. The changes of the seasons mean days get longer or shorter so to accurately predict the raising of the sun your clock must be reset. Plus for those who fly great distances we must reset according to the new environment and sadly suffer from jet lag while we are slowly adjusting (Johnson et al., 2008).

These clocks have evolved independently multiple times. In animals, plants, some fungi and recently discovered in cyanobacteria (Johnson et al., 2008). It was once thought because prokaryotes divide so raplidly that a 24 hour rhythm would be useless to them but that is not the case (Johnson et al., 1996). A great deal has been learnt about rhythms in bacteria but there are still many unanswered questions. The fact circadian clocks have evolved multiple times shows it offers a great advantage to those that posses one but was no present in the last common ancestor of life. Changing behaviour to only occur at a specific time of day is a great advantage to organisms, plants to photosynthesize and animals to look for food at the right times (Bell-Pedersen et al., 2005).

Cyanobacteria also rely on the sun for energy so predicting when the sun will rise allows them to start synthesising photosystem proteins before the sun raises while not producing them all night or waiting for the sun to raise. It has been shown when you make a mixed culture of bacteria with a mutant clock (no rhythmic changes in gene expression) and wild-type then wild-type will soon outcompete the mutants. Those that have a different length period (eg 16 hours rather than 24 hours) will always lose to wild-type in 24 hour day conditions but wild-type will lose in conditions that match the internal clock of these mutants (Yan et al., 1998). Interestingly in cyanobacteria the whole genome shows rhythmic expression! (Liu et al., 1995) This is in contrast to most organisms that control 15%-35% of their genome in a circadian dependant manner (McDonald and Rosbach, 2001; Correa et al., 2003; Michael and McClung, 2003). This is controlled by the supercoiled state of the whole genome! (Smith and Williams, 2006) I eagerly await the paper that explains how this achieved.

What is rather fascinating about this system is the oscillator. In most organisms gene expression is negatively regulated by its product in a way it cycles its levels over 24 hours and controls output (Bell-Pedersen et al., 2005). In cyanobacteria, this is not the case. Rhythmic expression of clock proteins occur but they do not repress there own synthesis. Also an in vitro oscillator can be setup. In the test tube the clock protein KiaC will cycle through different phosphorylation states if with KaiA, KaiB and ATP. In the organism this forms the post-translational oscillator (Nakajima et al., 2005). This is temperature compensatory and this is built into the ATPase activity of KaiC (Terauchi et al., 2007). It has been shown even when locked in one phosphorylation state you get a weak rhythm so a transcriptional-translational feedback loop as in eukaryotes is needed for a robust system (Kitayama et al., 2008) posing the question of whether this occurs in eukaryotes.

And to finish off I will add one final oddity about the circadian rhythms of cyanobacteria. Instead of using a photoreceptor to sense light inputs like other organisms they read the state of the metabolism. When light is high photosynthesis is occurring and the redox state of the cell changes and is seen as an input to reset the clock (Ivleva et al., 2005).

References

*Of importance
**Truly significant

Bell-Pedersen, D., Cassone, V.M., Earnest, D.J., Golden, S.S., Hardin, P.E., Thomas, T.L. and Zoran, M.J. (2005) Circadian rhythms from multiple oscillators: Lessons from diverse organisms. Nature Reviews Genetics, 6, 544-556.

Correa, A., Lewis, A.Z., Greene, A.V., March, I.J., Gomer, R.H. and Bell-Pedersen, D. (2003) Multiple oscillators regulate circadian gene expression in Neurospora. Proceedings of the National Academy of Sciences of the United States of America, 100, 13597-13602.

Ivleva, N.B., Bramlett, M.R., Lindahl, P.A. and Golden, S.S. (2005) LdpA: a component of the circadian clock senses redox state of the cell. Embo Journal, 24, 1202-1210.

Johnson, C.H., Golden, S.S., Ishiura, M. and Kondo, T. (1996) Circadian clocks in prokaryotes. Molecular Microbiology, 21, 5-11.

Johnson, C.H., Mori, T. and Xu, Y. (2008) A Cyanobacterial Circadian Clockwork. Current Biology, 18, R816-R825.

**Kitayama, Y., Nishiwaki, T., Terauchi, K. and Kondo, T. (2008) Dual KaiC-based oscillations constitute the circadian system of cyanobacteria. Genes & Development, 22, 1513-1521.

Liu, Y., Tsinoremas, N.F., Johnson, C.H., Lebedeva, N.V., Golden, S.S., Ishiura, M. and Kondo, T. (1995) Circadian Orchestration of Gene-Expression in Cyanobacteria. Genes & Development, 9, 1469-1478.

McDonald, M.J. and Rosbach, M. (2001) Microarray analysis and organization of circadian gene expression Drosophila. Cell, 107, 567-578.

Michael, T.P. and McClung, C.R. (2003) Enhancer trapping reveals widespread circadian clock transcriptional control in Arabidopsis. Plant Physiology, 132, 629-639.

*Nakajima, M., Imai, K., Ito, H., Nishiwaki, T., Murayama, Y., Iwasaki, H., Oyarna, T. and Kondo, T. (2005) Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science, 308, 414-415.

Smith, R.M. and Williams, S.B. (2006) Circadian rhythms in gene transcription imparted by chromosome compaction in the cyanobacterium Synechococcus elongatus. Proceedings of the National Academy of Sciences of the United States of America, 103, 8564-8569.

Terauchi, K., Kitayama, Y., Nishiwaki, T., Miwa, K., Murayama, Y., Oyama, T. and Kondo, T. (2007) ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria. Proceedings of the National Academy of Sciences of the United States of America, 104, 16377-16381.

Yan, O.Y., Andersson, C.R., Kondo, T., Golden, S.S. and Johnson, C.H. (1998) Resonating circadian clocks enhance fitness in cyanobacteria. Proceedings of the National Academy of Sciences of the United States of America, 95, 8660-8664.

The Life and Death of Elysia Chlorotica

Since the end of university I’ve had some trouble letting go of science as a constant occupation. Fortunately I was given the chance to post here about an old journal club presentation rather than go cold turkey, so here is a short piece on one of the most interesting organisms I’ve read about.

The picture on the left is of a sea slug of the genus Elysia which has evolved to blend in with its surroundings by looking almost exactly like a small leaf. The green colour that makes this camouflage so convincing is produced when the slug feeds on algae and steals their chloroplasts, subsequently storing them throughout its body. The use of chloroplast pigments to produce a leaf colour is common among sea slugs but the subject of this post is exceptional among the rest of its family. While in the majority of sea slugs the chloroplasts stop working within days or weeks of incorporation, those taken up by Elysia chlorotica can remain stable and active within the slug for at least ten months. As a result the slug is a rare example of a photosynthetic animal and it can survive without food for a long time, as long as it has light and carbon dioxide.

E. chlorotica has coevolved with a specific species of algae named Vaucheria litorea, which is a source of food and chloroplasts for the slug. The metamorphosis of E. chlorotica larvae into their slug form takes place only if the larvae are attached to filaments of V. litorea and in the absence of this algal species the larvae will die. Immediately after metamorphosis the slugs feed on the algae to which they are attached and take up their chloroplasts, incorporating them into their branched digestive tract.

As mentioned previously, most species of sea slug lose their chloroplast functions soon after obtaining them from algae. This is expected to be because the majority of genes required for chloroplast survival and function are encoded on the algal nucleus. Since animals do not have genes involved in chloroplast maintenance the question of how E. chlorotica manages to sustain its stolen plastids was investigated by several labs. In a recent study, the chloroplast genome of V. litorea was sequenced to determine whether it had greater genetic autonomy than previously sequenced chloroplasts, however, the chloroplast genome was found to be largely normal and was missing many genes essential for photosynthesis.

One of these missing genes, psbO, encodes a component of photosystem II which is vulnerable to damage during photosynthesis and needs to be regularly resynthesised for chloroplast function. Rumpho et al. were able to amplify the whole of this gene from sea slug DNA using primers based on seqeuence databases. The amplified sequence was shown to correspond exactly to the version of psbO from V. litorea, indicating that lateral gene transfer had taken place between the algae and the slug. The same sequence could be amplified from sea slug eggs which had not yet encountered algae, confirming that the sequence had entered the E. chlorotica germline. The authors behind this study speculate that all of the other genes needed for chloroplast survival and function have also been transferred to the slug germline, explaining the unique ability of E. chlorotica to maintain its chloroplasts for its entire life. So it seems that the sea slugs have stolen both DNA and organelles from algae, which is quite an achievement.

Another interesting property of E. chlorotica is that its generations are separated from each other. Whether slugs are collected in the lab or monitored in their natural environment, the entire adult population undergoes a synchronous death every year after they have laid their eggs. Pierce et al. observed that viral particles could be seen in the cytoplasm and nuclei of slugs just before their mass death and suggested that they may be responsible for the mortalities. The morphology of the viruses and the presence of reverse transcriptase activity suggest that they are retroviruses and, since the particles were found even under controlled laboratory conditions, it is likely that the virus is encoded on the genome of the sea slug. The group who made this observation suggested that the viruses may respond to annual environmental changes and kill the sea slugs in the spring, though this hypothesis has not been tested so far.

Unfortunately, the last paper I found on research into the viruses within the sea slugs was in 1999 and so research into this interesting aspect of the organism may not be ongoing. However I'm hoping to find some more about it in the future.

Rumpho, M. (2000). Solar-Powered Sea Slugs. Mollusc/Algal Chloroplast Symbiosis PLANT PHYSIOLOGY, 123 (1), 29-38 DOI: 10.1104/pp.123.1.29

Pierce, S., Maugel, T., Rumpho, M., Hanten, J., & Mondy, W. (1999). Annual Viral Expression in a Sea Slug Population: Life Cycle Control and Symbiotic Chloroplast Maintenance Biological Bulletin, 197 (1) DOI: 10.2307/1542990

Pierce, S., Massey, S., Hanten, J., & Curtis, N. (2003). Horizontal Transfer of Functional Nuclear Genes between Multicellular Organisms Biological Bulletin, 204 (3) DOI: 10.2307/1543594

Rumpho, M., Worful, J., Lee, J., Kannan, K., Tyler, M., Bhattacharya, D., Moustafa, A., & Manhart, J. (2008). From the Cover: Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica Proceedings of the National Academy of Sciences, 105 (46), 17867-17871 DOI: 10.1073/pnas.0804968105

Cell death, is it all about apoptosis?

Where would you be without cell death, well dead I reckon. Cell death is needed in development to shape you body, such as give you fingers by killing the webbing between them. Not only that, but cell death is needed to shape the development of the brain. These things are done by apoptosis, what I would describe and the clean way of dealing with dead cells. They get broken down by nice signals outside the cell or if something internal goes wrong they order themselves to die (like by p53 to stop the cell becoming a cancerous cell and killing you). When they break down they do it in such an ordered way you wouldn’t really notice it, the dead parts get eaten up by near by cells and no nasty lysomal enzymes get released to damage the rest of the tissue. However not all cells die in this lovely way. Necrosis is where the cell is damaged and really just bursts open and releases lysomal enzymes that damage the tissue and it causes an inflammatory response by releasing HMGB1 from the nucleus into the extracellular environment to clear away the corpses of the cells.

However the two may not be so distinct. The same injury can cause either and if the apoptosis mediators are not functioning through mutations then necrosis can take over. If ATP levels drop in a cell going through apoptosis necrosis kindly steps in and does the job. Necrosis seems to have a controlled cellular pathway involving ROS, calcium signalling and proteases being activated. Some of the necrosis program appears to work in the background of apoptosis and parts of apoptosis keep the death ordered. Necrosis also (sometimes) is beneficial to an organism. In rabbits it has been reported that excess cells in theur growth plates down by necrosis.

So necrosis may be damaging and causes a lot of brain damage in strokes and Alzheimer's disease, but it is probably a backup program for when apoptosis fails to handle the situation rather than simply the cell bursting open!

The figure is from: Leist M. and Jäättelä M. (2001) Four deaths and a funeral: From caspases to alternative mechanisms. Nature Reviews Molecular Cell Biology 2: 1-10.

'Green-turning-red spots' movie- world premiere

Once upon a time scientists didn't know how proteins where trafficked and changed along the Golgi apparatus. Some argued they were transported in vesicles from one type of cisterna to the other, where they were differently altered by different resident proteins. Others argued that the cisternae themselves were mobile and could become the next cisterna in sequence by the retrograde trafficking of the required enzymes... There was much pain and confusion, and the scientists were not happy. Until one day one group of scientists decided to see if little green dots on cells could become red. And they could. Everyone was happy, and they lived happily ever after...

--> The moment of truth- when green becomes red- and our understanding of life, the universe and everything changes...








As Dr Gareth Evans has put it, on the email that kindly provided the link to this movie:

'You can then get the popcorn in and enjoy the show! (obviously you'll also enjoy the fantastic educational benefits of a green spot changing to a red spot) '

http://www.nature.com/nature/journal/v441/n7096/extref/nature04717-s14.mov

For all of you poor children who have not been in his lectures (or who may have fallen asleep):

Basically a protein specific of cis cisterna (Man-6-P receptor) was labelled with GFP while one from trans cisterna (ARF proteins) was labelled with dsRed. The fact that a single cisterna changes colour indicates that are the enzymes that trafick between cisterna changing proteins, rather than proteins to be changed that trafick to different cisterna.

The original reference: Losev et al., (2006). Golgi maturation visualized in living yeast, Nature, 441. 1002-6

Osteoclasts are cool

Right, so this blog was needing a post... but as I should be writing an essay rather than playing with the computer it is a small one. Basically in a tutorial last week one of the guys talked about osteoclasts and ostebolasts. As you clever folks probably know, the osteoblasts are responsible by forming the bone while the osteoclasts eat it up. An equilibrium between the two is required, as excess of osteoclasts means less bone and an excess of osteoblasts means an extra-calcified bone- in any case, the bone becames brittle and more easily breaks. Any therapy to prevent osteoporosis is complicated by the fact that they normally tend to target osteoclasts... which besides eating up the bone also secreted components that promote osteoblast development, in order to guarantee the normal equilibrium (that allows bone renovation).

Really interesting, however, are the photographs below.

In this photo you can actually see the contact between an osteoclast. Osteoclasts produce hydrogen ions that acidify and dissolve the bone surface, as well as hydrolytic enzymes


My favourite one, which I cheesily baptized of osteoclast at breakfast, basically shows an osteoclast with some similarities to a snail- leaving behind not a trail of mucus but rather of eaten bone.

Finally, there is a movie of an osteoclast. Not extremelly interesting (it cannot beat the green-turning-to-red spots of Gareth Evans) but at least it shows that osteoclasts are motile (note that it has been speed up 100 times) and have multiple nuclei: http://www.brsoc.org.uk/gallery/movies/arnett_osteoclast2.mov