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.

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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.