Tag Archives: Early Career Researcher

How a motile cytoskeleton drives bacterial cell division

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seamusIn a recent issue of Science, the discovery of a key mechanism for bacterial cell division was reported. This work was carried out by Dr Seamus Holden’s lab (ICaMB) in collaboration with Professor Cees Dekker (TU Delft), Professor Yves Brun (Indiana University), Professor Mike VanNieuwenhze (Indiana University) and Professor Ethan Garner (Harvard University). Here, Seamus tells us about this discovery and what its implications could be for antimicrobial research.

Bacterial cell division is a lovely mechanistic problem in biology: how do the simplest living organisms build a crosswall at mid cell, against very high outwards pressure (think of a racing bike tyre), without bursting?  A ring of protein filaments forms around the future division site, and enzymes associated with this ring build a new crosswall that cleaves the bacteria in half. But what has remained completely mysterious is how these proteins work together as a single nanoscale machine to cut the bacterial balloon skin (cell wall) in two.

Cytoskeletal proteins FtsZ in live bacteria imaged in vertical nanocages

Cytoskeletal proteins FtsZ in live bacteria imaged in vertical nanocages

Working together with collaborators in Delft, Indiana and Harvard, we tracked the organization and motion of key division proteins as they build the dividing crosswall, and the organization of the newly built crosswall itself. We began by examining the motion of FtsZ, a cytoskeletal filament that is required for cell division – cytokinesis – in bacteria and is related to the tubulin cytoskeletal protein found in eukaryotic cells. Using high-resolution microscopy techniques, we found that FtsZ filaments move around the division site, traveling around the division ring. We imaged the motion of individual cell wall synthesis enzymes, and saw that the synthesis enzymes ride on FtsZ filaments, building new cell wall as they travel along the division site. This causes the cell wall to be synthesized in discrete sites that travel around the division site during cytokinesis, a process which we were able to observe directly by using dyes that label the bacterial cell wall. Using a variety of experimental techniques, we were able to speed up or slow down how fast FtsZ rotated around the cell. Strikingly, we found that the speed of FtsZ filament motion determines how fast the cell can divide. When FtsZ moves more rapidly, cell wall is produced more quickly, and cytokinesis happens faster. This shows that the motion of FtsZ is the critical overall controller of cell division.

One challenge that we faced was trying to look at the division proteins in actively dividing cells. At the earliest stages of division, it was possible to image division protein organization because the proteins in the partially assembled ring are sparsely distributed. However, a new strategy was required to measure how the dense protein network of actively dividing cells was organized. Normally, bacteria are immobilized flat on a microscope slide, and imaged from underneath, but unfortunately this places the division ring side-on, obscuring the motion and organization of division proteins. To solve this problem, we used nanofabrication technology, originally developed to manufacture computer chips, to create tiny gel nanocages to trap bacteria in an upright position.

Bacteria trapped in vertical nanocages

Bacteria trapped in vertical nanocages

By trapping individual bacteria upright, we were able to rotate the cell division ring so that it was fully visible on our high resolution microscope. This revealed the dynamic motion of FtsZ filaments as they travel around the entire division site:

Together, these results revealed the basic mechanistic principles of bacterial cell division: that the building of the division crosswall is orchestrated by moving cytoskeletal filaments.  Previously, the cytoskeleton was thought to serve as a static scaffold, recruiting other molecules and perhaps exerting some force to divide the cell. This new work demonstrates that all the components of cell division are in constant, controlled motion around the division site, driven by the fundamental dynamics of the cytoskeleton.

In the longer term, this study could open up novel antibiotic targets. Based on the discovery that the treadmilling motion of the bacterial cytoskeleton is critical for division, it may be possible to develop new drugs that specifically inhibit this motion, similar to how the chemotherapy drug taxol suppresses the motion of the cytoskeleton in cancer cells.

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Links

Explanatory animation: https://youtu.be/6dq2_gqKPfU (Animation credit TU Delft / Scixel)

Nanocage Video: nanocage-movie-2.

Science report: http://science.sciencemag.org/content/355/6326/739

Press release: http://bit.ly/2kwsnJf

Athena SWAN is open for discussion

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Posted by Suzanne Madgwick

Following on from the success of our Athena SWAN Bronze Award we began an open discussion on social media by posting an article about its principles and practice, the good and the bad! It’s been pretty clear that not everyone is entirely happy, some with the charter in principle and others with actions in ICaMB related to the charter. The issue is polarised both nationally and within the department. We certainly don’t want to shy away from this and so we asked you for your anonymous views.

The one consistent thread that emerged again from this feedback is that we all agree on the importance of gender equality in the workplace, this has never been in doubt. HOWEVER opinions on other aspects vary wildly, you’ll see from the following matrix that we don’t even all hold the same views on the AS team.

This has been a very productive exercise with which we can develop our future direction. Thank you for taking the time to post your comments. We’ve put together a team summary statement  and a full comments matrix to highlight our future approach and the types of conversations we are having. We have also organised the comments into a few main topics which are available by clicking on the pages below for a quick view.

We hope we can continue to openly discuss. So have a look at our statement for a more general view, the summaries focusing on the topics that interest you most and please do let us know what you think.

Positive discriminationPositive Discrimination

                                                         A Box ticking exerciseBox ticking 

 

Butterfly programmeThe Butterfly Program

 

                                                        BullyingAS4

 


AS5Having children harms your career

                                                        A historical problem?Historical problem?

 

Don’t forget to take the time to look through the full matrix. This is a working document that will continue to improve through open discussions and with your valuable feedback and help, so please leave your comments!

Thanks again

The AS team.

Not Athena SWAN again! The Good, the Bad and the Ugly

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In one of today’s dual posts, we get the personal opinion of Suzanne Madgwick, a research fellow in ICaMB, about her experiences and the pros and cons of Athena SWAN.

The following opinions are all my own and not necessarily those of ICaMB, other good opinions are available; no men or women were harmed during the preparation of this article.

4th of November 2013 was the first time I heard the term Athena SWAN. An email dropped into academic inboxes, a message which has no doubt been rolled out in one form or another across countless institutions throughout the country. Something along the lines of: ‘Inequalities between male and female academics which may exist need to be addressed ….. For many granting bodies this is becoming a major issue …….. NIHR have made it very clear that only institutions with at least an Athena Swan Silver Award will be eligible ……… others may follow …….. Self-Assessment Team …….. Application ……Volunteers’

?

         ?

Three thoughts ran through my head

Primarily confusion, in this position I can’t think of a time when I have felt discriminated against. Where has the notion that gender inequality exists in ICaMB come from? Whether I succeed or fail is based on many things; academic ability, resilience, character, free personal life choices and of course luck among others. I cannot currently identify a factor that could be singled out as a gender barrier. Sure, we work in a traditionally male-dominated environment but this is changing, gradually yes, but as far as I can see without conflict or resistance. Might it then be damaging to try and force this?

Secondly, why is there a possibility that government and charity money may in future only be awarded to institutions with a specific award? Is this necessarily the most responsible spend of money? When did the best research team stop being the one with the best idea? Given that there is increasing evidence to suggest that the most productive teams exist within flexible, progressive environments with good levels of female and male representation, again, are we not moving towards this anyway?

My final thought at the time was ‘Uh oh, I’m bound to be ‘asked’ to volunteer for our self-assessment team, this will be awkward’. But I reasoned that whilst I failed to see the existence of a problem I should help the department in an application. The Athena SWAN charter has us backed into a corner and whether I agree with it or not, in some way we will all benefit from an award.

Picture417 months on, I am now trapped in the frustrations of a Jekyll and Hyde type situation. I cannot ignore the fact that I still have these same objections and many more to boot. But I am also pleased to have become increasingly aware of the immense good that can come from a team striving to make improvements in relation to points 2 and 3 covered by the Athena SWAN charter.

The Good; in particular, but not limited to; mentoring schemes for both personal development and career progression, events for early career researchers to help identify and inform funding opportunities, promotion of flexible working hours, technical support and relief from additional duties for staff returning from leave, the formation of a team to identify, sponsor and encourage people who are able and talented but perhaps lack the self-promotion needed to reach the next level ……. and so on and so on. Brilliant! Everybody who has the ability and would like to, has an equal opportunity to stay in science. Creating a more flexible, inspirational working environment for all seems like a great idea, but continually lumping this together with ‘women’s issues’ is putting off a significant proportion of our workforce.

The Bad; nothing listed here is simply a gender issue, they are team issues and I am frustrated that all of these great positive changes are eclipsed by a much more visible yet awkward approach to addressing point 1.

Yes, there is evidence to suggest that women are sometimes a little more risk averse, less likely to put themselves forward for promotion, but this is by no means exclusive. If we have a mechanism in place to champion and support the different needs of all people, each and every time they need it, is this not equality without the need to keep using the word “women”? I can’t help thinking that there is a good dose of hypocrisy in all the ‘positive actions’ and events which are seen to be just for women. In the short term it’s generating friction and in the long term it certainly doesn’t seem like the best strategy when preaching fair play.

Picture1

Athena SWAN is suffering an image crisis. To the people who are not engaging in the initiative, I can’t blame you. I’m uncomfortable with the image of Athena SWAN and I would assume I’m supposed to be a benefactor. Despite all the good, we are alienating people; they assume it’s not for them, or like me, they don’t see the barrier. Can we consider for a moment that when we’re feeling energised and determined in our careers that it might be a little insulting to tell us we are being discriminated against and may need extra help? Prior to Athena SWAN I felt that my position was born of the factors that I have listed at the beginning of this article. Only now do I look around and wonder.

I’m beginning to get the feeling I have an ‘Athena SWAN’ label. I don’t want to highlight anybody in particular but I am not alone here. It doesn’t take much of an internet search to find high profile women making comments about feeling that recently they’ve been asked to speak more and more about women’s issues and less and less about science.

Of course women are different, 80% of us will have children and not even the power of Athena SWAN can switch over the uterus. But my children are my children, my choice, not a dent in my or my husband’s career. I have taken several years off; I am several years behind a peer who has not taken time out and this is as it should be. But, also as it should be, there were options available to me to return to science. I’m very pleased to report how well supported I have been in this, as I’m sure are the cohort of men who have also gained Career Re-Entry Fellowships.

The Ugly; the corridor murmuring. I’m not going to participate in anything to do with Athena SWAN but I’m going to moan about it anyway. But perhaps people feel they can’t speak up, the ugly side of political correctness. Please challenge us, we may agree with you. I am reminded of Hilary Lappin-Scott’s final phrase at last year’s equality in academia event “best use of all our talent”. Our self-assessment team is not balanced. We are getting lots of things right but we are also getting some things wrong, these are then the points that go noticed. We have certainly tried to concentrate on charter points two and three, but I for one feel very uncomfortable about the fact that we are hamstrung by the need to address all three.

The Leaky Pipeline; we can’t deny the ‘leaky pipeline,’ the drop off in the proportion of female scientists who progress from Postdoc to PI (though current ICaMB fellows are 47% female); the Athena SWAN initiative began with a need to address this. Nevertheless, we also can’t assume that we know all the reasons for the leak. Identifying these reasons is a big part of the challenge faced by the Athena SWAN self-assessment team. As crazy as it might sound, we do not all want to stay in science (though Bob if you are reading, I do). I have recently read that 88% of female PhD students do not want to stay in academia, but then neither do 79% of male PhD students. Surely through sponsorship, mentoring, flexible working etc., we can make sure that everybody who would like to stay in science has an equal opportunity based on merit, without making this an alienating gender issue.

This brings me back to our Athena Swan event last month where Professor Helen Arthur, Jill Golightly and Professor Melanie Welham all gave highly entertaining, outstanding talks about three very different very successful career paths. Sitting in my chair at the end of the afternoon I felt thoroughly inspired not because they are three inspirational women but because they are 3 inspirational people……. Only to then stand up and feel disheartened as turned and noticed the proportion of men in the audience. Have we done this? Has the Image of Athena SWAN has done this? With this in mind ……

AS

 

Athena SWAN – deconstructed

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In the second of today’s dual posts, we hear from Nancy Rios, Athena SWAN project officer for Newcastle University’s Faculty of Medical Sciences. Nancy explains why Athena SWAN is necessary, and how ICaMB aims to change its gender imbalance.

Athena SWAN – so what’s it all about? According to the tin, Athena SWAN is a charter set Bronze awardup by women’s networks to tackle the under-representation of women in STEMM. The model is simple – we analyse our local situation, evaluate our working practices and then develop strategies and actions to make the workplace fairer for everyone. Universities and their departments can apply for either a Bronze, Silver or Gold award (renewable every three years). ICaMB has recently been awarded a Bronze award. So far, so good.

Why Athena SWAN? Let’s start with the evidence.

In FMS, around 60% of undergraduate students are women.

Figure 1

 

In ICAMB, approximately 50% of PhD students, 40% of Postdocs, 50% of Fellows and 15% of permanent academic staff, including only 10% of Professors, are women.

Figure 2

Figure 3

These numbers aren’t unusual. In fact the alarming drop out rate and low proportion of women in senior, strategic positions is typical in STEMM departments in universities all over the country. So there’s the statistical evidence – women scientists aren’t progressing in academia at the same rate as men.

Lab picturesSo why does all this matter? Why make gender equality something that should be addressed in our workplace? Being concerned about the loss of women scientists isn’t about being politically correct and nor is it about feminism. Athena SWAN is about developing a competitive and effective workplace and making the most of all of our talent for the benefit of the University and for science. It’s about becoming a modern and dynamic employer that understands that women and men both become parents or carers and both make great scientists. Quality needs diversity. Recent research shows that teams and boards that include women make better decisions and perform better in business. That’s why diversity is a priority for our university.

OK, but why are so many women leaving? Maybe women just don’t want to stay in science. You can’t chain anyone to a bench and why would you want to? Actually, when you look at the evidence, the reasons that women leave are varied and complex. They lie in a combination of structural, cultural and systemic factors, both conscious and unconscious bias. Biology is a factor too, of course. Even though parental responsibilities can be shared, adding to the family just does have more immediate impact on the mother.

Bias may be a dirty word, but if we pretend it isn’t happening, we can’t do anything about it. In a very recent and rather hair-raising example of blatant bias a peer-reviewer suggested two female biologists get a man to co-author their paper to improve it.  Another more local example came from a researcher who was asked at a job interview (at a different university) whether or not she was planning children. She said she wasn’t, and the panel asked her how she knew. There is also the less obvious unconscious bias that we all inevitably have. Unconscious bias means that our behaviours and the decisions that we make are influenced without our knowing it by preconceptions that we’ve been developing since birth. Our brains develop short cuts that cause us to make assumptions and ignore objective facts. These shortcuts are natural and necessary for survival, but they are not so good for business. Evidence would suggest that unconscious bias can have an impact at work at any level you care to think about.

For example Moss-Racusin et al, 2012, found that professors (of both genders) when evaluating applications from students for a post of lab manager rated the ‘male’ applicant more competent and hirable, and offered a higher starting salary, than that of an otherwise identical ‘female’ applicant.

A 2014 study showed that when students were asked to rate teaching instructors of online courses they rated the male identity significantly higher than the female identity regardless of the actual gender.

And this bias can make a difference to which students are encouraged or ignored, who is asked to present group data at conferences or who is asked to make the tea. Biases can also make a difference to how much we feel we can achieve for ourselves.

There are also structural factors that can impact women more than men – out of date procedures and systems that make it hard to balance working with family life. For example, it tends to be more difficult for women to engage in networking opportunities, meetings and seminars after hours. Short term contracts, working abroad, travel, a ‘long hours’ culture and few opportunities to work part time are difficult for both men and women who have children or other dependants to look after.

It’s great to hear that researchers like Suzanne do not feel that they have personally experienced any discrimination. Unfortunately less positive accounts of women’s experiences at work are frequently heard. For example the PhD student who loved her field but wasn’t planning on staying beyond until her late twenties because she wanted to have a family and saw this as incompatible; an academic who got ill to the point where she was on medication trying to manage the transition back to work after maternity leave – she ‘didn’t dare’ ask for help; a professor in her 50s who pondered over whether the sacrifice she’d made twenty years earlier – she decided not to have children, in order to pursue her career – was really the right decision. There are accounts of bullying (gender related), discrimination (gender related) and resignation (in all senses of the word).

So what can Athena SWAN do?

SoapboxIn ICAMB, we’re using the Athena SWAN process to try and redress the balance and work towards a fairer workplace for all. For example, we looked at the first big attrition point for us – the transition from Postdoc to Lecturer. We found out that mentors were generally only available to Postdocs with fellowships, so we set up mentoring schemes to enable all Postdocs in FMS to access a mentor. We found that both women and men were unaware of what support they were entitled to around maternity and paternity leave and flexible working, so we’ve made an effort to highlight and publicise policies. We’ve committed to ensuring that all staff involved in recruitment panels attend training in unconscious bias. We provide additional time and money to support staff to attend training courses. We’ve started holding seminars at different times and we organise ‘equality in academia’ events. We are also engaging with national projects such as Soapbox Science.

Equality day

We are working with colleagues across FMS and the University on Athena SWAN. We are lobbying the University to provide a nursery with affordable creche provision for all staff. We’ve heard that many women have found it very difficult when returning from maternity leave to get their research back up to speed – so we’re in the process of setting up a programme that will offer research active staff (men and women) additional support at that time to make a successful transition back to a research career.

These are just a few of the small changes that we’ve started to make under Athena SWAN to make the workplace fairer for everyone, but it’s just the start of a journey. There are a plethora of other issues that are emerging that people are asking us to think about. What’s going on with our recruitment procedures that means we sometimes have so few women applying? How can people gain experience on committees? Can we simplify and update our policy around bringing children into the workplace in a fair but sensible way?

You may have noticed that the vast majority of actions benefit men as well as women, and ICAMBthere are changes that have a positive impact on everyone. That is why it is so important to get as many people as possible involved. We would love to have more volunteers on our Athena SWAN team to help out with input, ideas and challenges and help us achieve a Silver Award in the near future! Culture change is difficult and it’s inevitable that we won’t always get things right first time, so the more feedback we get, the better.

It’s concerning that Athena SWAN is so often misunderstood. There are ‘urban myths’ that it is about positive discrimination and giving women a ‘leg up’ the career ladder. If you think about it, this view is insulting to everyone. Let’s use the analogy of a footbal game – this isn’t about giving any player an advantage over another, its simply about levelling the pitch. We welcome an open discussion of the issues, of all the things that have been achieved so far and how they can be taken further.

Athena Swan team

Links

https://heidi.hesa.ac.uk/

http://news.sciencemag.org/scientific-community/2015/04/sexist-peer-review-elicits-furious-twitter-response

Moss-Racusin et al, 2012: http://www.pnas.org/content/109/41/16474

Online course rating: http://link.springer.com/article/10.1007%2Fs10755-014-9313-4

Soapbox Science: http://soapboxscience.org/

ECRs at ICaMB – Green transcription: how studying Cyanobacteria could change the world

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In the latest of our series on the Early Career Researchers of ICaMB we asked Dr Yulia Yuzenkova to tell us about her research and the route that took her from a PhD in Moscow to being awarded a Royal Society University Research Fellowship in Newcastle.

Yulia
Dr Yulia Yuzenkova

My early training was in Moscow, firstly as an undergraduate at Moscow State University and then for a PhD from the Russian Academy of Sciences. However, during my PhD I moved to the USA to study at the Waksman Institute (Rutgers University) with Prof Konstantin Severinov, where I was also a postdoc. My work in the laboratory was dedicated to the molecular mechanisms of inhibition of bacterial transcription by antibacterial peptides and small proteins from viruses. Transcription is the first step and critical regulatory checkpoint of gene expression. In all living organisms transcription is performed by multi-subunit RNA polymerases (RNAP). The central role of transcription in cellular metabolism and the presence of domains specific for bacteria make RNAP an obvious target for antibiotics. Yet, for decades, only one inhibitor of RNAP, rifampicin, has been used in the clinic to treat tuberculosis, while only recently, in 2011, lipiarmycin (fidaxomicin) was approved to eradicate Clostridium difficile. Two is a very small number, but this indicates that there is a good probability of finding new drugs that also work by targetting RNAP. Apart from their clinical significance, antibiotics and inhibitors of transcription in general have been proven to be very efficient molecular tools (see “RNAP details” below for more info).

During my second PostDoc in the lab of Prof Nikolay Zenkin in Newcastle, I have focused on the mechanisms controlling the fidelity of transcription. The copying of genetic information by RNAP is far from being absolutely precise, and RNAP whimsically dislikes reading some DNA sequences, resulting in ‘pauses’. RNAP is able to correct its own mistakes using a proofreading mechanism and the help of small proteins called transcript cleavage factors (explained in detail below). It seems to be important to have at least one cleavage factors; otherwise, the RNAP molecules stop, resulting in “traffic jams” as trailing molecules keep moving and bump into it.

Batch culturing of cyanobacteria

Batch culturing of cyanobacteria

It was therefore a big surprise for me to learn that one large group of bacteria, cyanobacteria (details below), do not encode anything even remotely resembling cleavage factors. I started to look for an explanation for this extraordinary fact. Are there any factors that might compensate for the absence of cleavage factors? Is cyanobacterial RNAP so accurate and at the same time processive that it does not need them? In searching for the answer, I realised that almost nothing is known about the molecular details of transcription in cyanobacteria, and so I decided to apply for a Royal Society University Research Fellowship to try to answer these questions.

Microscopy image of membrane-stained cell of Synechocystis sp 6803

Microscopy image of membrane-stained cell
of Synechocystis sp 6803

In performing preliminary experiments for my application, I became fascinated with cyanobacteria, as they are truly amazing organisms to work with. They are the only prokaryotes that exhibit the classic circadian clock, and are the bacteria with the most complex intracellular organisation. Moreover, they are one of the most ecologically important groups on Earth. They live everywhere where sunlight is available and produce 30% of atmospheric oxygen; some can even convert inert atmospheric nitrogen into a digestible form.

 

The 2D projection of the membrane structure of cyanobacterium reminds me of a labyrinth

The 2D projection of the membrane structure of cyanobacterium reminds me of a labyrinth

With my Royal Society University Research Fellowship I am planning to investigate the molecular details of the transcription machinery and will look for novel transcription factors that regulate this process. I am also going to test the metal requirements of cyanobacterial transcription, because metal composition of cyanobacterial cells is very different from other bacteria to suit the need of photosynthesis. Another fascinating question is how fast in the cyanobacterial cell, which is tightly packed with photosynthetic organelles, can molecules find their way through the membrane labyrinth.

 

 

The nitty-gritty science:

RNAP in detail:

Inhibitors of RNAP as molecular tools for understanding its functions. A wide range of targets of known inhibitors is mapped on the structure of bacterial RNAP. We contributed to understanding the modes of action of inhibitors marked in bold.

Inhibitors of RNAP as molecular tools for understanding its functions. A wide range of targets of known inhibitors is mapped on the structure of bacterial RNAP. We contributed to understanding the modes of action of inhibitors marked in bold.

Studying the antibiotics and inhibitors modes of action have helped us and other groups to discover previously unknown functions and structural domains of RNA polymerase. For example, work on rifampicin shed light on geometry of the RNA exit path, long before the crystal structure of RNAP was solved. Moreover, streptolydigin led to the discovery of the novel catalytic domain, while microcinJ25 confirmed the proposed entry channel for substrates and tagetitoxin provided insight into the mechanisms of RNAP translocation along the template.

Newly synthesised RNA participates in the proofreading  in a ribozyme-like manner. This method of proofreading, probably a remnant from the distant past, is extremely slow. To accelerate proofreading (and to escape from pauses), all 3 domains of life encode non-homologous, but very similarly folded, small proteins called transcript cleavage factors. In E.coli, GreA is an example of a protein that fulfils this role. Some bacteria have several homologs of GreA (in E.coli there are at least 6). It seems to be important to have at least one, because if these cleavage factors are depleted in the cell, the RNAP molecules on the actively transcribed genes stop, obstructing transcription, but also the chromosomal replication machinery moving along the same DNA.

Cyanobacteria:

Cyanobacteria have been hailed as future photobioreactors.  Indeed, when supplied with little more than tap water and light, engineered cyanobacteria can produce all sorts of compounds from sunscreen to biofuels. Cyanobacteria can also be used for environmental applications such as greenhouse gas fixing and cleaning water from industrial pollutants. These initiatives, however, are compromised by slow growth of cyanobacteria and limited knowledge of their basic biology. By putting more effort into research, the potential abilities of cyanobacteria can eventually be harnessed on the industrial level. With this we could make a giant leap towards a future “greener” economy. With a little bit of imagination, it is not hard to envisage cyanobacteria helping humanity to colonise new worlds, and to permit them to inhabit the first lunar and martial greenhouses in not so distant future.

Dr Yulia Yuzenkova’s ICaMB website: http://www.ncl.ac.uk/camb/staff/profile/yulia.yuzenkova

Royal Society University Research Fellowship: https://royalsociety.org/grants/schemes/university-research/

ECRs at ICaMB: RNA Quality Control

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Claudia SchneiderIn the latest of our series focussing on the ECRs in ICaMB, we feature Dr Claudia Schneider. Claudia obtained her PhD from the Philipps-University in Marburg, Germany. She then moved to the UK to work with Prof David Tollervey at the Wellcome Trust Centre for Cell Biology in Edinburgh. In 2011, she was awarded a Royal Society University Research Fellowship and started her own lab at ICaMB. Here, Claudia describes her research, and how being alarmed during her postgraduate studies triggered her long-term research interest.

By Dr Claudia Schneider

Hi, my name is Claudia Schneider, and my Royal Society University Research Fellowship has allowed me to set up my own group here at ICaMB to study enzymes involved in RNA processing and quality control.

I am originally from Germany, where I did my undergraduate studies and my PhD. Many people might think that Germany is the land of cars or lederhosen – but in truth it is really the land of bread (and beer!). It might therefore not come as a surprise that baker’s yeast has become my favourite model organism.

Click on the image to find out how to make these budding buns!

Click on the image to find out how to make these budding buns!

During my undergraduate studies I was first introduced to RNA and I was (and am still) amazed by its many known and still emerging functions in the cell. We now know that almost the entire eukaryotic genome is transcribed, but only a small fraction of the transcripts are protein-coding messenger RNAs (mRNAs). The others are stable and unstable non-coding RNAs (ncRNAs), which are involved in all aspects of gene expression. RNA molecules are often extensively processed before they’re functional, and each processing step is subject to quality control mechanisms. If you want to know more about the life and death of non-coding RNAs, have a look at this recent review.

Yeast has not always been my first choice to study RNA metabolism, since the object of my PhD project in Prof Reinhard Lührmann’s lab turned out to be completely missing in baker’s yeast. Back then I worked on nuclear pre-mRNA splicing, the removal of non-coding introns from precursor mRNAs catalysed by the spliceosome. It was an exciting time in the splicing field: A second low abundance “minor” spliceosome had just been discovered in most multicellular eukaryotes (with the strange and still not readily explainable exception of C. elegans), and this complex is not present in yeast. The minor spliceosome recognises a rare class of introns (<0.5%) with different consensus sequences at the splice sites and has since been linked to a number of human diseases. During my PhD, I purified and biochemically characterised the snRNP components of this unusual pre-mRNA splicing machinery in human and Drosophila cells.

Since then, I have been fascinated by biochemical and enzymatic assays involving RNA such as in vitro splicing assays, where in vitro transcribed pre-mRNAs are mixed with purified spliceosomes. The goal of such an experiment is to observe precise and (hopefully) pretty intron removal in the test tube – but, to my great annoyance, success was every so often hampered by a powerful ribonuclease (RNase) contamination in the assay that completely trashed the precious RNA substrates. Generic and aggressive RNases like RNase A are found on our skin, are incredibly stable and can even survive boiling.

Common decoration on lab surfaces during my PhD

Common decoration on lab surfaces during my PhD

It is therefore fair to say that my scientific career was majorly influenced by constant warnings by my mentor, who told me that all RNases are evil and must be destroyed. However, for my postdoc, I decided to face my fears and look these evil RNases in the eye, in the humble model system yeast. During my time with Prof David Tollervey at the University of Edinburgh, I learned that there are many different types of RNases, and only very few of them are promiscuous and chop RNA to bits.

The majority of RNases are very sophisticated and versatile enzymes. Several RNases are capable of degrading only specific RNA molecules, or only function under certain circumstances, and protein co-factors often assist in substrate recognition. RNases are crucial elements in RNA quality control or surveillance systems, which distinguish aberrant from “normal” RNA molecules. One clinically important RNA surveillance pathway is called “nonsense-mediated decay” or NMD, and this system recognises and degrades a specific class of defective mRNAs to limit the synthesis of truncated and potentially toxic proteins. NMD defects are linked to ~30% of all inherited human diseases (e.g. Duchenne muscular dystrophy and forms of b-thalassemia) as well as certain types of cancer. In addition to quality control/surveillance, where RNAs are mostly completely degraded, a growing number of RNases have been shown to be responsible for precise processing or “trimming” of precursor RNA molecules to produce their functional forms.

Exonucleases were long believed to be the main players involved in RNA recognition and processing/turnover. However, this model was recently challenged by the identification of endonucleases containing PIN (PilT N-terminus) domains, which appear to play key roles in RNA metabolism. Eight PIN domain proteins and therefore putative endonucleases are encoded in the genome of budding yeast, and this includes three largely uncharacterised “orphan” nucleases.

Overall it is still puzzling to me how individual RNases “make the decision” to either completely degrade or carefully process a specific RNA. Given the ever-growing number of non-coding transcripts in the cell, I am also keen to know which RNases are responsible for which substrates and what the so-far uncharacterised putative PIN domain endonucleases in yeast are doing!

To this end, our lab is using an RNA-protein cross-linking method called “CRAC” (UV cross-linking and analysis of cDNA) to identify the targets of PIN domain endonucleases on a transcriptome-wide scale. The CRAC method and the machinery to cross-link yeast cultures were developed by Sander Granneman at the University of Edinburgh, when we were both PostDocs in Prof David Tollervey’s lab. Sander now has his own lab too, and he runs a CRAC-blog. Interestingly, the cross-linking device we are using was originally designed to sterilise sewage water, but it is now also commercially available for research. With this setup, yeast cells are cross-linked while they are growing in culture, which is crucial to identify the often very transient interactions between nucleases and their target RNAs. This system provides a huge advantage over more traditional cross-linkers like the “Stratalinker”, which requires pelleting and cooling the cells on ice before cross-linking. It is, however, also much bigger and takes up a whole bench in the lab – but I guess there is a drawback to everything! In any case: if you want to find RNA targets for your favourite yeast protein – get in touch!!

Cultures of Saccharomyces cerevisiae can be “zapped”, while they are growing: It only takes 100 seconds!

Cultures of Saccharomyces cerevisiae can be “zapped”, while they are growing: It only takes 100 seconds!

Transcriptome-wide RNA-protein interaction analyses generate huge datasets and we use RNA binding and nuclease assays, as well as co-precipitation studies, to validate the in vivo cross-linking results for individual PIN domain endonucleases.

With the help of an ERASMUS exchange student, Franziska Weichmann, who spent 6 months in my lab last year, we have made good progress with two putative PIN domain endonucleases that are linked to ribosome biogenesis. We were able to identify their binding sites on the pre-ribosomal RNAs, as well as co-factors that are important to recruit them into the pre-ribosome. We have also set up an in vitro system with recombinant proteins, and we are currently trying to convince one of them to specifically cleave its proposed rRNA substrate in the test tube – and we are slowly getting there…..

Like the other ICaMB ECRs, who posted on this Blog before, I would like to finish by saying that having my own lab has been an exciting and (on most days) enjoyable adventure so far – and I am looking forward to the next set of challenges…

ECRs at ICaMB: Copying the blueprint of life – Understanding DNA replication

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Heath MurrayIn the latest of our series focussing on the Early Career Researchers (ECRs) in ICaMB, we feature Dr Heath Murray.  After completing undergraduate studies at the University of California, Los Angeles and then obtaining his Ph.D. from the University of Wisconsin-Madison, Heath came to the UK to join the lab of Prof Jeff Errington in Oxford. From there he re-located to ICaMB, and in 2009 was awarded a Royal Society University Research Fellowship. Here, Heath describes his research into the mechanisms of DNA replication, and explains why he became interested in this field.

By Dr Heath Murray

Hello, my name is Heath Murray and I’m a Royal Society University Research Fellow in ICaMB’s Centre for Bacterial Cell Biology (CBCB) studying DNA replication. DNA is one of the most important molecules required for life because it encodes the information, or the blueprint, used to build a cell (i.e. the most basic unit of an organism). In order for a cell to create new cells it must synthesize an exact copy of its DNA, an extraordinary process when you consider that the genomes of most cells contain millions of individual DNA subunits!

Bacillus subtilis is a useful model system as it proliferates rapidly and is amenable to genetic, cell biological, biochemical, and structural analyses

Bacillus subtilis is a useful model system as it proliferates rapidly and is amenable to genetic, cell biological, biochemical, and structural analyses

Bacteria are ideal model systems to study this fundamental process because they are much less complex than human cells (e.g. all of their DNA is encoded by a single chromosome, whereas humans have 23), and this allows us to understand how they work at the greatest possible level of detail.

I was introduced to bacteria when I was an undergraduate student and the effect was transformative. My mentors taught me how to add a specific gene (a DNA sequence) to a bacterial cell, and if it worked properly then the bacteria would turn blue!

Bacterial colonies turn blue if they contain a gene that degrades specific sugars.

Bacterial colonies turn blue if they contain a gene that degrades specific sugars.

That basic genetic experiment was one of the coolest things I had ever done, and from that point on I worked hard to learn the trade of “bacterial genetics”.

Today my research group focuses on understanding how DNA replication is controlled so that each new cell will end up containing an exact copy of the genetic material from its predecessor. We employ a wide range of complementary experimental techniques: genetic engineering of bacterial strains, biochemical analysis of purified proteins, and fluorescence microscopy.

In the hot room to check my plates.... I haven't even stopped to take my jacket or backpack off yet!

In the hot room to check my plates…. I haven’t even stopped to take my jacket or backpack off yet!

Fluorescence microscopy is a particular strength of the CBCB because there are several bespoke systems specifically designed for bacterial cells (bacteria are 10-100 times smaller than most human cells). One of the core approaches we use is to genetically engineer a protein we want to study so that it will be fused to a special reporter protein called GFP (Green Fluorescent Protein, originally isolated from jellyfish!) within the cell. Using this approach we can then visualize where our test protein is because it fluoresces when exposed to a specific wavelength of light. Some of our microscopes are so sophisticated that we can observe the location of single proteins and track their movements within living cells.

At the bench.

At the bench.

One of the approaches we often use is to visualize specific regions of the genome within living cells. First, a specific DNA binding protein ( called “LacI”) is genetically fused to GFP. Second, the DNA sequence recognized by LacI (called “lacO”) can be genetically integrated into any location of the genome. Since I study DNA replication, I am particularly interested in the site of the bacterial chromosome where DNA synthesis is initiated (called the “replication origin”). Third, fluorescent dyes are added to cells that bind to the cell membrane and the DNA. Finally, we utilize our fluorescent microscopes to visualize the location of replication origins within individual cells. In the image shown, the live bacterial cells contain chromosomes that are in the process of being replicated, and therefore they have duplicated and separated their replication origins!! This image also emphasizes the fact that although bacteria lack the organelles found in eukaryotic cells, they are nonetheless highly organized (notice how the replication origins are characteristically located at the outer edge of each chromosome).

The GFP protein from jellyfish can be used to fluorescently tag proteins in vivo. Fluorescence microscopy can then be used to localise the tagged protein within the bacterial cell.

The GFP protein from jellyfish can be used to fluorescently tag proteins in vivo. Fluorescence microscopy can then be used to localise the tagged protein within the bacterial cell.

Well that’s it for my first ICaMB blog! I hope you enjoyed hearing about how I became interested in bacterial genetics and about my work on bacterial DNA replication. Please feel welcome to contact me if you have any questions or if you would like further information regarding my research.

 

Links

Royal Society URF http://royalsociety.org/grants/schemes/university-research/

Centre for Bacterial Cell Biology (CBCB) http://www.ncl.ac.uk/cbcb/

 

ECRs at ICaMB: Solving 3D puzzles

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by Dr Paula Salgado

After nearly one year editing the ICaMBlog, the time has come for me to tell you about my science and work since I joined ICaMB almost 18 months ago.

The fact that it has been 18 months since I moved up North to establish my own research group seems to have snuck up on me… Don’t get me wrong, so much has happened that, if anything, it’s surprising it all took place in 1 and a half years. At the same time, the feeling of a new adventure is still there.

Science is a constant adventure to seek new knowledge, to understand new mechanism, to see new things. In my case, to see into the very core of life’s machines: proteins. I use X-ray protein crystallography to probe the structure of proteins. It’s a bit like solving a puzzle: fitting the pieces of information together until we have a 3D view of the protein.

It is actually fitting that my blog post is the first ICaMB publishes in 2014 as this is the International Year of Crystallography. I could write a lot about it, but for now, I’ll leave you with an amazing video made by the Royal Institution that explains it all – in cartoons! If you want to know more about Crystallography, the Ri has a great collection of videos there, including Prof Stephen Curry’s Friday Evening Discourse, which I strongly recommend.

Freezing protein crystals for data collection at Diamond Light Source. H&S warning: liquid nitrogen is a hazard and we do handle it safely. At this point, I was just dipping the crystals into a small volume, all other procedures handling larger volumes involve wearing appropriate protection.

As a protein crystallographer, I’ve always been interested in proteins that have a relevance to human disease and used this technique to understand their structure and function. In the last few years, I’ve worked on proteins from human pathogens associated with hospital acquired infections, particularly Clostridium difficile and Candida albicans. However, protein structures don’t necessarily give us all the answers and they must be complemented with biochemical studies, as well as in vivo experiments. So my long term goal has become to establish a Structural Microbiology group, where we focus on structure determination of key proteins and complexes involved in pathogenicity as well as on their functional in vivo characterisation.

This is a challenge as it means stepping out of my structural biology comfort zone into the world of microbiology and cell biology. Not that I haven’t stepped out of my comfort zone before – if anything, those are areas that featured strongly during my undergraduate training as a Biochemistry student at the University of Porto in Portugal. In those days, choosing to do protein crystallography as my undergraduate project was the big step into the unknown. A trend that continued as a post-doc, when I joined Dr Cota and Prof Mathews group, a Nuclear Magnetic Resonance (NMR) lab at Imperial College, learning a completely different approach to protein structure determination. And just before coming to ICaMB, I worked in Prof Fairweather’s microbiology lab and always tried to learn a bit about the techniques others were using. So the current idea of bringing structural biology and microbiology expertise together in my lab is the natural evolution of these experiences.

C. difficile cells (green rods) lining the microvilli of the human gut. © Wellcome Trust (CC-BY)

Since joining ICaMB, I’ve focused on 2 main projects, both involving proteins from C. difficile. This spore forming strict anaerobe is resistant to most antibiotics and colonises the gut of individuals whose microbiome has been disturbed by these drugs. It is the most prevalent cause of gastrointestinal infections in hospitals and is a major cause of morbidity and mortality in the hospital environment. Despite recent decreases in the number of deaths and infections as hygiene procedures have improved in the UK, over 1600 people died in England and Wales in 2012 due to C. difficile infections (CDI). It also causes a huge burden to health systems, with an estimated €3,000 million per annum costs in the EU.

C. difficile disease symptoms are caused by the toxins it releases in a process that has been extensively studied over the years. However, the mechanisms of colonisation of the gut and spore formation are poorly understood. So we have been focusing on proteins involved in these two mechanisms.

Firstly, I’ve been trying to determine the structure of SlpA, the main protein constituent in C. difficile S-layer. S-layer is a paracrystalline coat that covers the cell and is presumed to act like a defense mechanism, as well as being involved in gut colonisation. This work, initiated a few years ago in Prof Fairweather’s lab is now a joint collaboration between our two labs and Dr Fagan, at Sheffield University.

SlpA crystals viewed under polarised light (protein crystals are birefringent, unlike salt crystals)

 

 

As this protein has tendency to form 2D paracrystalline layers, getting well ordered 3D crystals required for X-ray crystallography has been a challenge, but I have now succeeded in obtaining good crystals. However, other hurdles still need to be overcome to get a structure – but we are getting there!

 

 

Our lab: Adam Crawshaw and Paula Salgado

Last year, Adam Crawshaw joined my group as a BBSRC Doctoral Training Programme (DTP) student and we started a new project, looking at a complex between two membrane proteins that are essential for spore formation. As spores are the infectious agents, revealing the molecular details sporulation is important to understand the pathogenicity and infection cycle of C. difficile.

SpoIIQ and SpoIIIAH localise at the membranes of the forming forespore. Green: Membrane; Red: SNAP-tagged proteins.

When spores are first formed, a small cell (forespore) is engulfed by the larger mother cell, physically isolating it from the environment and nutrients in the medium. So, for the forespore to fully mature, it needs a nurturing channel to the mother cell. The two proteins we are studying  – SpoIIQ from the forespore membrane and SpoIIIAH from the mother cell membrane – create this channel. We have already successfully produced recombinant versions of the proteins and shown their interaction in vitro. In collaboration with Prof Henriques at ITQB, Lisbon, we also established their localisation during C. difficile spore formation. Next: crystals! But we are also investigating potential enzymatic activity both in vitro and in vivo, to bring the structure and biology together.

It has been an exciting year and a half – a steep learning curve with many new tasks, from supervising students to managing a lab and teaching undergraduates and postgraduates. The adventure continues, with new challenges and exciting discoveries ahead.

 

Links

International Year of Crystallography http://www.iycr2014.org/

Royal Institution Crystallography gallery http://richannel.org/celebrating-crystallography

Office for National Statistics (Clostridium difficile data) http://www.ons.gov.uk/ons/rel/subnational-health2/deaths-involving-clostridium-difficile/2012/index.html

European Centre for Disease Intervention and Control on Clostridium difficile  http://www.ecdc.europa.eu/en/healthtopics/healthcare-associated_infections/clostridium_difficile_infection/pages/index.aspx

BBSRC Doctoral Training Programme http://www.ncl.ac.uk/fms/dtp/