Ferrous: new paper on FerriTagging proteins in cells

We have a new paper out. It’s not exactly news, because the paper has been up on bioRxiv since December 2016 and hasn’t changed too much. All of the work was done by Nick Clarke when he was a PhD student in the lab. This post is to explain our new paper to a general audience.

The paper in a nutshell

We have invented a new way to tag proteins in living cells so that you can see them by light microscopy and by electron microscopy.

Why would you want to do that?

Proteins do almost all of the jobs in cells that scientists want to study. We can learn a lot about how proteins work by simply watching them down the microscope. We want to know their precise location. Light microscopy means that the cells are alive and we can watch the proteins move around. It’s a great method but it has low resolution, so seeing a protein’s precise location is not possible. We can overcome this limitation by using electron microscopy. This gives us higher resolution, but the proteins are stuck in one location. When we correlate images from one microscope to the other, we can watch proteins move and then look at them with high resolution. All we need is a way to see the proteins so that they can be seen in both types of microscope. We do this with tagging.

Tagging proteins so that we can see them by light microscopy is easy. A widely used method is to use a fluorescent protein such as GFP. We can’t see GFP in the electron microscope (EM) so we need another method. Again, there are several tags available but they all have drawbacks. They are not precise enough, or they don’t work on single proteins. So we came up with a new one and fused it with a fluorescent protein.

What is your EM tag?

We call it FerriTag. It is based on Ferritin which is a large protein shell that cells use to store iron. Because iron scatters electrons, this protein shell can be seen by EM as a particle. There was a problem though. If Ferritin is fused to a protein, we end up with a mush. So, we changed Ferritin so that it could be attached to the protein of interest by using a drug. This meant that we could put the FerriTag onto the protein we want to image in a few seconds. In the picture on the right you can see how this works to FerriTag clathrin, a component of vesicles in cells.

We can watch the tagging process happening in cells before looking by EM. The movie on the right shows green spots (clathrin-coated pits in a living cell) turning orange/yellow when we do FerriTagging. The cool thing about FerriTag is that it is genetically encoded. That means that we get the cell to make the tag itself and we don’t have to put it in from outside which would damage the cell.

What can you use FerriTag for?

Well, it can be used to tag many proteins in cells. We wanted to precisely localise a protein called HIP1R which links clathrin-coated pits to the cytoskeleton. We FerriTagged HIP1R and carried out what we call “contextual nanoscale mapping”. This is just a fancy way of saying that we could find the FerriTagged HIP1R and map where it is relative to the clathrin-coated pit. This allowed us to see that HIP1R is found at the pit and surrounding membrane. We could even see small changes in the shape of HIP1R in the different locations.

We’re using FerriTag for lots of projects. Our motivation to make FerriTag was so that we could look at proteins that are important for cell division and this is what we are doing now.

Is the work freely available?

Yes! The paper is available here under CC-BY licence. All of the code we wrote to analyse the data and run computer simulations is available here. All of the plasmids needed to do FerriTagging are available from Addgene (a non-profit company, there is a small fee) so that anyone can use them in the lab to FerriTag their favourite protein.

How long did it take to do this project?

Nick worked for four years on this project. Our first attempt at using ribosomes to tag proteins failed, but Nick then managed to get Ferritin working as a tag. This paper has broken our lab record for longest publication delay from first submission to final publication. The diagram below tells the whole saga.

 

The publication process was frustratingly slow. It took a few months to write the paper and then we submitted to the first journal after Christmas 2016. We got a rapid desk rejection and sent the paper to another journal and it went out for review. We had two positive referees and one negative one, but we felt we could address the comments and checked with the journal who said that they would consider a revised paper as an appeal. We did some work and resubmitted the paper. Almost six months after first submission the paper was rejected, but with the offer of a rapid (ha!) publication at Nature Communications using the peer review file from the other journal.

Hindsight is a wonderful thing but I now regret agreeing to transfer the paper to Nature Communications. It was far from rapid. They drafted in a new reviewer who came with a list of new questions, as well as being slow to respond. Sure, a huge chunk of the delay was caused by us doing revision experiments (the revisions took longer than they should because Nick defended his PhD, was working on other projects and also became a parent). However, the journal was really slow. The Editor assigned to our paper left the journal which didn’t help and the reviewer they drafted in was slow to respond each time (6 and 7 weeks, respectively). Particularly at the end, after the paper was ‘accepted in principle’ it took them three weeks to actually accept the paper (seemingly a week to figure out what a bib file is and another to ask us something about chi-squared tests). Then a further three weeks to send us the proofs, and then another three weeks until publication. You can see from the graphic that we sent back the paper in the third week of February and only incurred a 9-day delay ourselves, yet the paper was not published until July.

Did the paper improve as a result of this process? Yes and no. We actually added some things in the first revision cycle (for Journal #2) that got removed in subsequent peer review cycles! And the message in the final paper is exactly the same as the version on bioRxiv, posted 18 months previously. So in that sense, no it didn’t. It wasn’t all a total waste of time though, the extra reviewer convinced us to add some new analysis which made the paper more convincing in the end. Was this worth an 18-month delay? You can download our paper and the preprint and judge for yourself.

Were we unlucky with this slow experience? Maybe, but I know other authors who’ve had similar (and worse) experiences at this journal. As described in a previous post, the publication lag times are getting longer at Nature Communications. This suggests that our lengthy wait is not unique.

There’s lots to like about this journal:

  • It is open access.
  • It has the Nature branding (which, like it or not, impresses many people).
  • Peer review file is available
  • The papers look great (in print and online).

But there are downsides too.

  • The APC for each paper is £3300 ($5200). Obviously open access must cost something, but there a cheaper OA journals available (albeit without the Nature branding).
  • Ironically, paying a premium for this reputation is complicated since the journal covers a wide range of science and its kudos varies depending on subfield.
  • It’s also slow, and especially so when you consider that papers have often transferred here from somewhere else.
  • It’s essentially a mega journal, so your paper doesn’t get the same exposure as it would in a community-focused journal.
  • There’s the whole ReadCube/SpringerNature thing…

Overall it was a negative publication experience with this paper. Transferring a paper along with the peer review file to another journal has worked out well for us recently and has been rapid, but not this time. Please leave a comment particularly if you’ve had a positive experience and redress the balance.

The post title comes from “Ferrous” by Circle from their album Meronia.

Pentagrammarspin: why twelve pentagons?

This post has been in my drafts folder for a while. With the World Cup here, it’s time to post it!

It’s a rule that a 3D assembly of hexagons must have at least twelve pentagons in order to be a closed polyhedral shape. This post takes a look at why this is true.

First, some examples from nature. The stinkhorn fungus Clathrus ruber, has a largely hexagonal layout, with pentagons inserted. The core of HIV has to contain twelve pentagons (shown in red, in this image from the Briggs group) amongst many hexagonal units. My personal favourite, the clathrin cage, can assemble into many buckminsterfullerene-like shapes, but all must contain at least twelve pentagons with a variable number of hexagons.

The case of clathrin is particularly interesting because clathrin triskelia can assemble into a flat hexagonal lattice on membranes. If clathrin is going to coat a vesicle, that means 12 pentagons need to be introduced. So there needs to be quite a bit of rearrangement in order to do this.

You can see the same rule in everyday objects. The best example is a football, or soccer ball, if you are reading in the USA.

The classic design of football has precisely twelve pentagons and twenty hexagonal panels. The roadsign for football stadia here in the UK shows a weirdly distorted hexagonal array that has no pentagons. 22,543 people signed a petition to pressurise the authorities to change it, but the Government responded that it was too costly to correct this geometrical error.

So why do all of these assemblies have 12 pentagons?

In the classic text “On Growth and Form” by D’Arcy Wentworth Thompson, polyhedral forms in nature are explored in some detail. In the wonderfully titled On Concretions, Specules etc. section, the author notes polyhedral forms in natural objects.

One example is Dorataspis, shown left. The layout is identical to the D6 hexagonal barrel assembly of a clathrin cage shown above. There is a belt of six hexagons, one at the top, one at the bottom (eight total) and twelve pentagons between the hexagons. In the book, there is an explanation of the maths behind why there must be twelve pentagons in such assemblies, but it’s obfuscated in bizarre footnotes in latin. I’ll attempt to explain it below.

To shed some light on this we need the help of Euler’s formulae. The surface of a polyhedron in 3D is composed of faces, edges and vertices. If we think back to the football the faces are the pentagons and hexgonal panels, the edges are the stitching where two panels meet and the vertices are where three edges come together. We can denote faces, edges and vertices as f, e and v, respectively. These are 2D, 1D and zero-dimensional objects, respectively. Euler’s formula which is true for all polyhedra is:

\(f – e + v = 2\)

If you think about a cube, it has six faces. It has 12 edges and 8 vertices. So, 6 – 12 + 8 = 2. We can also check out a the football above. This has 32 faces (twelve pentagons, twenty hexagons), 90 edges and sixty vertices. 32 – 90 + 60 = 2. Feel free to check it with other polyhedra!

Euler found a second formula which is true for polyhedra where three edges come together at a vertex.

\(\sum (6-n)f_{n} = 12\)

in this formula, \(f_{n}\) means number of n-gons.

So let’s say we have dodecahedron, which is a polyhedron made of 12 pentagons. So \(n\) = 5 and \(f_{n}\) = 12, and you can see that \((6-5)12 = 12\).

Let’s take a more complicated object, like the football. Now we have:

\(((6-6)20) + ((6-5)12) = 12\)

You can now see why the twelve pentagons are needed. Because 6-6 = 0, we can add as many hexagons as we like, this will add nothing to the left hand side. As long as the twelve pentagons are there, we will have a polyhedron. Without them we don’t. This is the answer to why there must be twelve pentagons in a closed polyhedral assembly.

So how did Euler get to the second equation? You might have spotted this yourself for the f, e, v values for the football. Did you notice that the ratio of edges to vertices is 3:2? This is because each edge has two vertices at either end (it is a 1D object) and remember we are dealing with polyhedra with three edges at each vertex. so \(v = \frac{2}{3}e\). Also, each edge is at the boundary of two polygons. So \( e = \frac{1}{2}\sum n f_{n}\). You can check that with the values for the cube or football above. We know that \(f = \sum f_{n}\), this just means that the number of faces is the sum of all the faces of all n-gons. This means that:

\(f – e + v = 2\)

Can be turned into

\(f – (1/3)e = \sum n f_{n} – \frac{1}{6}\sum n f_{n} = 2\)

Let’s multiply by 6 to get, oh yes

\(\sum (6-n)f_{n} = 12\)

There are some topics for further exploration here:

  • You can add 0, 2 or 10000 hexagons to 12 pentagons to make a polyhedron, but can you add just one?
  • What happens when you add a few heptagons into the array?

Image credits (free-to-use/wiki or):

Clathrus ruber – tineye search didn’t find source.

HIV cores – Briggs Group

Exploded football – Quora

The post title comes from “Pentagrammarspin” by Steve Hillage from the 2006 remaster of his LP Fish Rising

Parallel lines: new paper on modelling mitotic microtubules in 3D

We have a new paper out! You can access it here.

The people

This paper really was a team effort. Faye Nixon and Tom Honnor are joint-first authors. Faye did most of the experimental work in the final months of her PhD and Tom came up with the idea for the mathematical modelling and helped to rewrite our analysis method in R. Other people helped in lots of ways. George did extra segmentation, rendering and movie making. Nick helped during the revisions of the paper. Ali helped to image samples… the list is quite long.

The paper in a nutshell

We used a 3D imaging technique called SBF-SEM to see microtubules in dividing cells, then used computers to describe their organisation.

What’s SBF-SEM?

Serial block face scanning electron microscopy. This method allows us to take an image of a cell and then remove a tiny slice, take another image and so on. We then have a pile of images which covers the entire cell. Next we need to put them back together and make some sense of them.

How do you do that?

We use a computer to track where all the microtubules are in the cell. In dividing cells – in mitosis – the microtubules are in the form of a mitotic spindle. This is a machine that the cell builds to share the chromosomes to the two new cells. It’s very important that this process goes right. If it fails, mistakes can lead to diseases such as cancer. Before we started, it wasn’t known whether SBF-SEM had the power to see microtubules, but we show in this paper that it is possible.

We can see lots of other cool things inside the cell too like chromosomes, kinetochores, mitochondria, membranes. We made many interesting observations in the paper, although the focus was on the microtubules.

So you can see all the microtubules, what’s interesting about that?

The interesting thing is that our resolution is really good, and is at a large scale. This means we can determine the direction of all the microtubules in the spindle and use this for understanding how well the microtubules are organised. Previous work had suggested that proteins whose expression is altered in cancer cause changes in the organisation of spindle microtubules. Our computational methods allowed us to test these ideas for the first time.

Resolution at a large scale, what does that mean?

The spindle is made of thousands of microtubules. With a normal light microscope, we can see the spindle but we can’t tell individual microtubules apart. There are improvements in light microscopy (called super-resolution) but even with those improvements, right in the body of the spindle it is still not possible to resolve individual microtubules. SBF-SEM can do this. It doesn’t have the best resolution available though. A method called Electron Tomography has much higher resolution. However, to image microtubules at this large scale (meaning for one whole spindle), it would take months or years of effort! SBF-SEM takes a few hours. Our resolution is better than light microscopy, worse than electron tomography, but because we can see the whole spindle and image more samples, it has huge benefits.

What mathematical modelling did you do?

Cells are beautiful things but they are far from perfect. The microtubules in a mitotic spindle follow a pattern, but don’t do so exactly. So what we did was to create a “virtual spindle” where each microtubule had been made perfect. It was a bit like “photoshopping” the cell. Instead of straightening the noses of actresses, we corrected the path of every microtubule. How much photoshopping was needed told us how imperfect the microtubule’s direction was. This measure – which was a readout of microtubule “wonkiness” – could be done on thousands of microtubules and tell us whether cancer-associated proteins really cause the microtubules to lose organisation.

The publication process

The paper is published in Journal of Cell Science and it was a great experience. Last November, we put up a preprint on this work and left it up for a few weeks. We got some great feedback and modified the paper a bit before submitting it to a journal. One reviewer gave us a long list of useful comments that we needed to address. However, the other two reviewers didn’t think our paper was a big enough breakthrough for that journal. Our paper was rejected*. This can happen sometimes and it is frustrating as an author because it is difficult for anybody to judge which papers will go on to make an impact and which ones won’t. One of the two reviewers thought that because the resolution of SBF-SEM is lower than electron tomography, our paper was not good enough. The other one thought that because SBF-SEM will not surpass light microscopy as an imaging method (really!**) and because EM cannot be done live (the cells have to be fixed), it was not enough of a breakthrough. As I explained above, the power is that SBF-SEM is between these two methods. Somehow, the referees weren’t convinced. We did some more work, revised the paper, and sent it to J Cell Sci.

J Cell Sci is a great journal which is published by Company of Biologists, a not-for-profit organisation who put a lot of money back into cell biology in the UK. They are preprint friendly, they allow the submission of papers in any format, and most importantly, they have a fast-track*** option. This allowed me to send on the reviews we had and including our response to them. They sent the paper back to the reviewer who had a list of useful comments and they were happy with the changes we made. It was accepted just 18 days after we sent it in and it was online 8 days later. I’m really pleased with the whole publishing experience with J Cell Sci.

 

* I’m writing about this because we all have papers rejected. There’s no shame in that at all. Moreover, it’s obvious from the dates on the preprint and on the JCS paper that our manuscript was rejected from another journal first.

** Anyone who knows something about microscopy will find this amusing and/or ridiculous.

*** Fast-track is offered by lots of journals nowadays. It allows authors to send in a paper that has been reviewed elsewhere with the peer review file. How the paper has been revised in light of those comments is assessed by at the Editor and one peer reviewer.

Parallel lines is of course the title of the seminal Blondie LP. I have used this title before for a blog post, but it matches the topic so well.

Reaching Out

Outreach means trying to engage the public with what we are doing in our research group. For me, this mainly means talking to non-specialists about our work and showing them around the lab. These non-specialists are typically interested members of the public and mainly supporters of the charity that funds work in my lab (Cancer Research UK). The most recent batch of activities have prompted this post on doing outreach.

The challenge

Outreach is challenging. Taking part in these events made me realise what a tough job it is to do science communication, and how good the best the communicators are.

There are many ways that an outreach talk is tougher to give than a research seminar. Not least because explaining what we do in the lab can quickly spiral down into a full-on Cell Biology 101 lecture.

A statement like “we work on process x and we are studying a protein called y”, needs to be followed by “jobs in cells are done by proteins”, then maybe “proteins are encoded by genes”, in our DNA, which is a bunch of letters, oh there’s mRNA, ahhh stop! Pretty soon, it can get too confusing for the audience. In a seminar, the level of knowledge is already there, so protein x can be mentioned without worrying about why or how it got there.

On the other hand, giving an outreach talk is much easier than giving a seminar because the audience is already warm to you and they don’t want you to stuff it up. It’s a bit like giving a speech at a wedding.

The challenge is exciting because it means that our work needs to be explained plainly and placed in a bigger context. If you get the chance to explain your work to a lay audience, I recommend you try.

Disarming questions

The big difference between doing a scientific talk for scientists and talking to non-specialists is in the questions. They can be disarming, for various reasons. Here are a few that I have had on recent visits. How would you answer?

Can you tell the difference [down the microscope] between cells from a black person versus those from a white person!?

For context, we had just looked at some HeLa cells down the microscope and I had explained a little bit about Henrietta Lacks and the ethical issues surrounding this cell line.

You mentioned evolution but I think you’ll find that the human cell is just too intricate. How do you think cells are really made?

Hint: it doesn’t matter what you reply. You will be unlikely to change their mind.

Do you dream of being famous? What will be your big discovery?

I’ve also been asked “are we close to a cure for cancer?”. It’s important to temper people’s enthusiasm here I think.

Are you anything to do with [The Crick]? No? Good! It’s a waste of money and it shouldn’t have been built in London!

I had wondered if lay people knew about The Crick, which is now the biggest research institute in the UK. Clearly they have! I tried to explain that The Crick is a chance to merge several institutes that already existed in London and so it would save money on running these places.

Aren’t you just being exploited by the pharmaceutical industry?

This person was concerned that academics generate knowledge which is then commercialised by companies.

My friend took a herbal remedy and it cured his cancer. Why aren’t you working on that?

Like the question rejecting evolution, it is difficult for people to abandon their N-of-one/anecdotal knowledge.

Does X cause cancer?

This is a problem of the media in our country I think. Who seem to be on a mission to categorise everything (red meat, wine, tin foil) into either cancer-causing or cancer-preventing.

As you can see, the questions are wide-ranging, which is unsettling in itself. It’s very different to “have you tried mutating serine 552 to test if the effect is one of general negative charge on the protein?” that you get in a research seminar.

The charity that organises some of the events I’ve been involved in are really supportive and give a list of good ways to answer “typical questions”. However, most questions I get are atypical, and the anticipated questions about animal research or embryo cloning do not arise.

I find it difficult to give a succinct answer to these lay questions. I try to give an accurate reply, but this leads to  long and complicated answer that probably confuses the person even more. I have the same problem with children’s questions, which often get me scurrying to Wikipedia to find the exact answer for “why the sky is blue”. I should learn to just give a vaguely correct answer and not worry about the details so much.

Amazing questions

The best questions are those where you can tell that the person has really got into it. In the last talk I gave, I described “stop” and “go” signals for cell division. One person asked

How does a cell suddenly know that it has to divide? It must get a signal from somewhere… what is that signal?

My initial reply was that asking these sorts of questions is what doing science is all about!

Two more amazing questions:

Is it true that scientists are secretive with their results and think more about advancing their careers than publicising their findings openly to give us value for money?

This was from a supporter of the charity who had read a piece in The Guardian about scientific publishing. She followed up by asking why do scientists put their research behind paywalls. I found this tough to answer because I suddenly felt responsible for the behaviour of the entire scientific community.

You mentioned taxol and the side effects. I was taking that for my breast cancer and it is true what you said. It was very painful and I had to stop treatment.

This was the first time a patient had talked to me about their experience of things that were actually in my talk. This was a stark reminder that the research I am doing is not as abstract as I think. It also made me more cautious about the way I talk about current treatments, since people in the room may be actually taking them!

Good support

With the charity I’ve been to Polo Clubs, hotels, country houses, Bishop’s houses, relay events in public parks. The best part is welcoming people to our lab. These might be a Mayor or people connected wth the city football team, but mainly they are interested supporters of the charity. It’s nice to be able to explain where their money goes and what a life in cancer research is really like.

To do these events, there is a team of people doing all the organisation: inviting participants, sorting out parking, tea and coffee etc. The team are super-enthusiastic and they are really skilled at talking to the public. The events could not go ahead without them. So, a big thank you to them. I’ve also been helped by the folks in the lab and colleagues in my building who have helped to show visitors around and let them see cells down the microscope etc.

Give it a try

Of course there are many other ways to engage the public in our research. This is just focussed on talking to non-scientists and the issues that arise. As I’ve tried to outline here, it’s a fun challenge. If you get the opportunity to do this, give it a try.

The post title comes from “Reaching Out” by Matthew Sweet from his Altered Beast LP. Lovely use of diminished seventh in a pop song and of course the drums are by none other than Mick Fleetwood.

Pull Together: our new paper on “The Mesh”

We have a new paper out! You can access it here.

Title of the paper: The mesh is a network of microtubule connectors that stabilizes individual kinetochore fibers of the mitotic spindle

bundle1What’s it about? When a cell divides, the two new cells need to get the right number of chromosomes. If this process goes wrong, it is a disaster which may lead to disease e.g. cancer. The cell shares the chromosomes using a “mitotic spindle”. This is a tiny machine made of microtubules and other proteins. We have found that the microtubules are held together by something called “the mesh”. This is a weblike structure which connects the microtubules and gives them structural support.

Does this have anything to do with cancer? Some human cancer cells have high levels of  proteins called TACC3 and Aurora A kinase. We know that TACC3 is changed by Aurora A kinase. This changed form of TACC3 is part of the mesh. In our paper we mimic the cancer condition by increasing TACC3 levels. The mesh changes and the microtubules become wonky. This causes problems for dividing cells. It might be possible to target TACC3 using drugs to treat certain types of cancer, but this is a long way in the future.

Who did the work? Faye Nixon, a PhD student in the lab did most of the work. She used a method to look at mitotic spindles in 3D to study the mesh. My lab actually discovered the mesh by accident. A previous student, Dan Booth – back in 2011 – was looking at mitotic spindles to try and get 3D electron microscopy (tomography) working in the lab. Tomography works just like a CAT scan in a hospital, but on a much smaller scale. The mesh is found in the gaps between microtubules that are 25 nanometre wide (1 nanometre is 1 billionth of a metre), this is about 3,000 times smaller than a human hair, so it is very small! It was Dan who found the mesh and gave it the name. Other people in the lab did some really nice work which helped us to understand how the mesh works in dividing cells. Cristina Gutiérrez-Caballero did some experiments using a different type of microscope and Fiona Hood contributed some test tube experiments. Ian Prior at University of Liverpool, co-supervises Faye and helped with electron microscopy.

Have you discovered a new structure in cells? Yes and No. All cell biologists dream of finding a new structure in cells. It’s so unlikely though. Scientists have been looking at cells since the 17th Century and so the chances of seeing something that no-one has seen before are very small. In the 1970s, “inter-microtubule bridges” in the mitotic spindle were described using 2D electron microscopy. What we have done is to look at these structures in 3D for the first time and find that they are a network rather than individual connectors.

The work was funded by Cancer Research UK and North West Cancer Research Fund.

References

Nixon, F.M., Gutiérrez-Caballero, C., Hood, F.E., Booth, D.G., Prior, I.A. & Royle, S.J. (2015) The mesh is a network of microtubule connectors that stabilizes individual kinetochore fibers of the mitotic spindle eLife, doi: 10.7554/eLife.07635

This post is written in plain English to try to describe what is in the paper. I’m planning on writing a more technical post on some of the spatial statistics we developed as part of this paper.

The post title is from “Pull Together” a track from Shack’s H.M.S. Fable album.

Zero Tolerance

We were asked to write a Preview piece for Developmental Cell. Two interesting papers which deal with the insertion of amphipathic helices in membranes to influence membrane curvature during endocytosis were scheduled for publication and the journal wanted some “front matter” to promote them.

Our Preview is paywalled – sorry about that – but I can briefly tell you why these two papers are worth a read.

The first paper – a collaboration between EMBL scientists led by Marko Kaksonen – deals with the yeast proteins Ent1 and Sla2. Ent1 has an ENTH domain and Sla2 has an ANTH domain. ENTH stands for Epsin N-terminal homology whereas ANTH means AP180 N-terminal homology. These two domains are known to bind membrane and in the case of ENTH to tubulate and vesiculate giant unilamellar vesicles (GUVs). Ent1 does this via an amphipathic helix “Helix 0” that inserts into the outer leaflet to bend the membrane. The new paper shows that Ent1 and Sla2 can bind together (regulated by PIP2) and that ANTH regulates ENTH so that it doesn’t make lots of vesicles, instead the two team up to make regular membrane tubules. The tubules are decorated with a regular “coat” of these adaptor proteins. This coat could prepattern the clathrin lattice. Also, because Sla2 links to actin, then actin can presumably pull on this lattice to help drive the formation of a new vesicle. The regular spacing might distribute the forces evenly over large expanses of membrane.

The second paper – from David Owen’s lab at CIMR in Cambridge – shows that CALM (a protein with an ANTH domain) actually has a secret Helix 0! They show that this forms on contact with lipid. CALM influences the size of clathrin-coated pits and vesicles, by influencing curvature. They propose a model where cargo size needs to be matched to vesicle size, simply due to the energetics of pit formation. The idea is that cells do this by regulating the ratio of AP2 to CALM.

You can read our preview and the papers by Skruzny et al and Miller et al in the latest issue of Dev Cell.

The post title and the title of our Preview is taken from “Zero Tolerance” by Death from their Symbolic LP. I didn’t want to be outdone by these Swedish scientists who have been using Bob Dylan song titles and lyrics in their papers for years.

Sticky End

We have a new paper out! You can access it here.

The work was mainly done by Cristina Gutiérrez Caballero, a post-doc in the lab. We had some help from Selena Burgess and Richard Bayliss at the University of Leicester, with whom we have an ongoing collaboration.

The paper in a nutshell

We found that TACC3 binds the plus-ends of microtubules via an interaction with ch-TOG. So TACC3 is a +TIP.

What is a +TIP?

EBTACCMitotic
EB3 (red) and TACC3 (green) at the tips of microtubules in mitotic spindle

This is a term used to describe proteins that bind to the plus-ends of microtubules. Microtubules are a major component of the cell’s cytoskeleton. They are polymers of alpha/beta-tubulin that grow and shrink, a feature known as dynamic instability. A microtubule has polarity, the fast growing end is known as the plus-end, and the slower growing end is referred to as the minus-end. There are many proteins that bind to the plus-end and these are termed +TIPs.

OK, so what are TACC3 and ch-TOG?

They are two proteins found on the mitotic spindle. TACC3 is an acronym for transforming acidic coiled-coil protein 3, and ch-TOG stands for colonic hepatic tumour overexpressed gene. As you can tell from the names they were discovered due to their altered expression in certain human cancers. TACC3 is a well-known substrate for Aurora A kinase, which is an enzyme that is often amplified in cancer. The ch-TOG protein is thought to be a microtubule polymerase, i.e. an enzyme that helps microtubules grow. In the paper, we describe how TACC3 and ch-TOG stick together at the microtubule end. TACC3 and ch-TOG are at the very end of the microtubule, they move ahead of other +TIPs like “end-binding proteins”, e.g. EB3.

What is the function of TACC3 as a +TIP?

We think that TACC3 is piggybacking on ch-TOG while it is acting as a polymerase, but any biological function or consequence of this piggybacking was difficult to detect. We couldn’t see any clear effect on microtubule dynamics when we removed or overexpressed TACC3. We did find that loss of TACC3 affects how cells migrate, but this is not likely to be due to a change in microtubule dynamics.

I thought TACC3 and ch-TOG were centrosomal proteins…

In the paper we look again at this and find that there are different pools of TACC3, ch-TOG and clathrin (alone and in combination) and describe how they reside in different places in the cell. Although ch-TOG is clearly at centrosomes, we don’t find TACC3 at centrosomes, although it is on microtubules that cluster near the centrosomes at the spindle pole. TACC3 is often described as a centrosomal protein in lots of other papers, but this is quite misleading.

What else?

NeonCellWe were on the cover – whatever that means in the digital age! We imaged a cell expressing tagged EB3 proteins, EB3 is another +TIP. We coloured consecutive frames different colours and the result looked pretty striking. Biology Open picked it as their cover, which we were really pleased about. Our paper is AOP at the moment and so hopefully they won’t change their mind by the time it appears in the next issue.

Preprinting

This is the second paper that we have deposited as a preprint at bioRxiv (not counting a third paper that we preprinted after it was accepted). I was keen to preprint this particular paper because we became aware that two other groups had similar results following a meeting last summer. Strangely, a week or so after preprinting and submitting to a journal, a paper from a completely different group appeared with a very similar finding! We’d been “scooped”. They had found that the Xenopus homologue of TACC3 was a +TIP in retinal neuronal cultures. The other group had clearly beaten us to it, having submitted their paper some time before our preprint. The reviewers of our paper complained that our data was no longer novel and our paper was rejected. This was annoying because there were lots of novel findings in our paper that weren’t in theirs (and vice versa). The reviewers did make some other constructive suggestions that we incorporated into the manuscript. We updated our preprint and then submitted to Biology Open. One advantage of the preprinting process is that the changes we made can be seen by all. Biology Open were great and took a decision based on our comments from the other journal and the changes we had made in response to them. Their decision to provisionally accept the paper was made in four days. Like our last experience publishing in Biology Open, it was very positive.

References

Gutiérrez-Caballero, C., Burgess, S.G., Bayliss, R. & Royle, S.J. (2015) TACC3-ch-TOG track the growing tips of microtubules independently of clathrin and Aurora-A phosphorylation. Biol. Open doi:10.1242/​bio.201410843.

Nwagbara, B. U., Faris, A. E., Bearce, E. A., Erdogan, B., Ebbert, P. T., Evans, M. F., Rutherford, E. L., Enzenbacher, T. B. and Lowery, L. A. (2014) TACC3 is a microtubule plus end-tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types. Mol. Biol. Cell 25, 3350-3362.

The post title is taken from the last track on The Orb’s U.F.Orb album.

Outer Limits

This post is about a paper that was recently published. It was the result of a nice collaboration between me and Francisco López-Murcia and Artur Llobet in Barcelona.

The paper in a nutshell
The availability of clathrin sets a limit for presynaptic function

Background
Clathrin is a three legged protein that forms a cage around membranes during endoctosis. One site of intense clathrin-mediated endocytosis (CME) is the presynaptic terminal. Here, synaptic vesicles need to be recaptured after fusion and CME is the main route of retrieval. Clathrin is highly abundant in all cells and it is generally thought of as limitless for the formation of multiple clathrin-coated structures. Is this really true? In a neuron where there is a lot of endocytic activity, maybe the limits are tested?
It is known that strong stimulation of neurons causes synaptic depression – a form of reversible synaptic plasticity where the neuron can only evoke a weak postsynaptic response afterwards. Is depression a vesicle supply problem?

What did we find?
We showed that clathrin availability drops during stimulation that evokes depression. The drop in availability is due to clathrin forming vesicles and moving away from the synapse. We mimicked this by RNAi, dropping the clathrin levels and looking at synaptic responses. We found that when the clathrin levels drop, synaptic responses become very small. We noticed that fewer vesicles are able to be formed and those that do form are smaller. Interestingly, the amount of neurotransmitter (acetylcholine) in the vesicles was much less than the volume of the vesicles as measured by electron microscopy. This suggests there is an additional sorting problem in cells with lower clathrin levels.

Killer experiment
A third reviewer was called in (due to a split decision between Reviewers 1 and 2). He/she asked a killer question: all of our data could be due to an off-target effect of RNAi, could we do a rescue experiment? We spent many weeks to get the rescue experiment to work, but a second viral infection was too much for the cells and engineering a virus to express clathrin was very difficult. The referee also said: if clathrin levels set a limit for synaptic function, why don’t you just express more clathrin? Well, we would if we could! But this gave us an idea… why don’t we just put clathrin in the pipette and let it diffuse out to the synapses and rescue the RNAi phenotype over time? We did it – and to our surprise – it worked! The neurons went from an inhibited state to wild-type function in about 20 min. We then realised we could use the same method on normal neurons to boost clathrin levels at the synapse and protect against synaptic depression. This also worked! These killer experiments were a great addition to the paper and are a good example of peer review improving the paper.

People
Fran and Artur did almost all the experimental work. I did a bit of molecular biology and clathrin purification. Artur and I wrote the paper and put the figures together – lots of skype and dropbox activity.
Artur is a physiologist and his lab like to tackle problems that are experimentally very challenging – work that my lab wouldn’t dare to do – he’s the perfect collaborator. I have known Artur for years. We were postdocs in the same lab at the LMB in the early 2000s. We tried a collaborative project to inhibit dynamin function in adrenal chromaffin cells at that time, but it didn’t work out. We have stayed in touch and this is our first paper together. The situation in Spain for scientific research is currently very bad and it deteriorated while the project was ongoing. This has been very sad to hear about, but fortunately we were able to finish this project and we hope to work together more in the future.

We were on the cover!
25.coverNow the scientific literature is online, this doesn’t mean so much anymore, but they picked our picture for the cover. It is a single cell microculture expressing GFP that was stained for synaptic markers and clathrin. I changed the channels around for artistic effect.

What else?
J Neurosci is slightly different to other journals that I’ve published in recently (my only other J Neurosci paper was published in 2002). For the following reasons:

  1. No supplementary information. The journal did away with this years ago to re-introduce some sanity in the peer review process. This didn’t affect our paper very much. We had a movie of clathrin movement that would have gone into the SI at another journal, but we simply removed it here.
  2. ORCIDs for authors are published with the paper. This gives the reader access to all your professional information and distinguishes authors with similar names. I think this is a good idea.
  3. Submission fee. All manuscripts are subject to a submission fee. I believe this is to defray the costs of editorial work. I think this makes sense, although I’m not sure how I would feel if our paper had been rejected.

Reference:

López-Murcia, F.J., Royle, S.J. & Llobet, A. (2014) Presynaptic clathrin levels are a limiting factor for synaptic transmission J. Neurosci., 34: 8618-8629. doi: 10.1523/JNEUROSCI.5081-13.2014

Pubmed | Paper

The post title is taken from “Outer Limits” a 7″ Single by Sleep ∞ Over released in 2010.