Methods papers for MD997

I am now running a new module for masters students, MD997. The aim is to introduce the class to a range of advanced research methods and to get them to think about how to formulate their own research question(s).

The module is built around a paper which is allocated in the first session. I had to come up with a list of methods-type papers, which I am posting below. There are 16 students and I picked 23 papers. I aimed to cover their interests, which are biological but with some chemistry, physics and programming thrown in. The papers are a bit imaging-biased but I tried to get some ‘omics and neuro in there. There were some preprints on the list to make sure I covered the latest stuff.

The students picked their top 3 papers and we managed to assign them without too much trouble. Every paper got at least one vote. Some papers were in high demand. Fitzpatrick et al. on cryoEM of Alzheimer’s samples and the organoid paper from Lancaster et al. had by far the most votes.

The students present this paper to the class and also use it to formulate their own research proposal. Which one would you pick?

  1. Booth, D.G. et al. (2016) 3D-CLEM Reveals that a Major Portion of Mitotic Chromosomes Is Not Chromatin Mol Cell 64, 790-802. http://dx.doi.org/10.1016/j.molcel.2016.10.009
  2. Chai, H. et al. (2017) Neural Circuit-Specialized Astrocytes: Transcriptomic, Proteomic, Morphological, and Functional Evidence Neuron 95, 531-549 e9. http://dx.doi.org/10.1016/j.neuron.2017.06.029
  3. Chang, J.B. et al. (2017) Iterative expansion microscopy Nat Methods 14, 593-599. http://dx.doi.org/10.1038/nmeth.4261
  4. Chen, B.C. et al. (2014) Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution Science 346, 1257998. http://dx.doi.org/10.1126/science.1257998
  5. Chung, K. & Deisseroth, K. (2013) CLARITY for mapping the nervous system Nat Methods 10, 508-13. http://dx.doi.org/10.1038/nmeth.2481
  6. Eichler, K. et al. (2017) The Complete Connectome Of A Learning And Memory Center In An Insect Brain bioRxiv. http://dx.doi.org/10.1101/141762
  7. Fitzpatrick, A.W.P. et al. (2017) Cryo-EM structures of tau filaments from Alzheimer’s disease Nature 547, 185-190. http://dx.doi.org/10.1038/nature23002
  8. Habib, N. et al. (2017) Massively parallel single-nucleus RNA-seq with DroNc-seq Nat Methods 14, 955-958. http://dx.doi.org/10.1038/nmeth.4407
  9. Hardman, G. et al. (2017) Extensive non-canonical phosphorylation in human cells revealed using strong-anion exchange-mediated phosphoproteomics bioRxiv. http://dx.doi.org/10.1101/202820
  10. Herzik, M.A., Jr. et al. (2017) Achieving better-than-3-A resolution by single-particle cryo-EM at 200 keV Nat Methods. http://dx.doi.org/10.1038/nmeth.4461
  11. Jacquemet, G. et al. (2017) FiloQuant reveals increased filopodia density during breast cancer progression J Cell Biol 216, 3387-3403. http://dx.doi.org/10.1083/jcb.201704045
  12. Jungmann, R. et al. (2014) Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT Nat Methods 11, 313-8. http://dx.doi.org/10.1038/nmeth.2835
  13. Kim, D.I. et al. (2016) An improved smaller biotin ligase for BioID proximity labeling Mol Biol Cell 27, 1188-96. http://dx.doi.org/10.1091/mbc.E15-12-0844
  14. Lancaster, M.A. et al. (2013) Cerebral organoids model human brain development and microcephaly Nature 501, 373-9. http://dx.doi.org/10.1038/nature12517
  15. Madisen, L. et al. (2012) A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing Nat Neurosci 15, 793-802. http://dx.doi.org/10.1038/nn.3078
  16. Penn, A.C. et al. (2017) Hippocampal LTP and contextual learning require surface diffusion of AMPA receptors Nature 549, 384-388. http://dx.doi.org/10.1038/nature23658
  17. Qin, P. et al. (2017) Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9 Nat Commun 8, 14725. http://dx.doi.org/10.1038/ncomms14725
  18. Quick, J. et al. (2016) Real-time, portable genome sequencing for Ebola surveillance Nature 530, 228-232. http://dx.doi.org/10.1038/nature16996
  19. Ries, J. et al. (2012) A simple, versatile method for GFP-based super-resolution microscopy via nanobodies Nat Methods 9, 582-4. http://dx.doi.org/10.1038/nmeth.1991
  20. Rogerson, D.T. et al. (2015) Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog Nat Chem Biol 11, 496-503. http://dx.doi.org/10.1038/nchembio.1823
  21. Russell, M.R. et al. (2017) 3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy J Cell Sci 130, 278-291. http://dx.doi.org/10.1242/jcs.188433
  22. Strickland, D. et al. (2012) TULIPs: tunable, light-controlled interacting protein tags for cell biology Nat Methods 9, 379-84. http://dx.doi.org/10.1038/nmeth.1904
  23. Yang, J. et al. (2015) The I-TASSER Suite: protein structure and function prediction Nat Methods 12, 7-8. http://dx.doi.org/10.1038/nmeth.3213

If you are going to do a similar exercise, Twitter is invaluable for suggestions for papers. None of the students complained that they couldn’t find three papers which matched their interests. I set up a slide carousel in Powerpoint with the front page of each paper together with some key words to tell the class quickly what the paper was about. I gave them some discussion time and then collated their choices on the board. Assigning the papers was quite straightforward, trying to honour the first choices as far as possible. Having an excess of papers prevented too much horse trading for the papers that multiple people had picked.

Hopefully you find this list useful. I was inspired by Raphaël posting his own list here.

Start Me Up: Endocytosis on demand

We have a new paper out. The title is New tools for ‘hot-wiring’ clathrin-mediated endocytosis with temporal and spatial precision. You can read it here.

Cells have a plasma membrane which is the barrier between the cell’s interior and the outside world. In order to import material from outside, cells have a special process called endocytosis. During endocytosis, cells form a tiny bubble of plasma membrane and pull it inside – taking with it a little pocket of the outside world. This process is very important to the cell. For example, it is one way that cells import nutrients to live. It also controls cell movement, growth, and how cells talk to one another. Because it is so important, cell biologists have studied how endocytosis works for decades.

Studying endocytosis is tricky. Like naughty children, cells simply do not do what they are told. There is no way to make a cell in the lab “do endocytosis”. It does it all the time, but we don’t know when or where on the cell surface a vesicle will be made. Not only that, but when a vesicle is made, we don’t really know what cargo it contains. It would be helpful to cell biologists if we could bring cells under control. This paper shows a way to do this. We demonstrate that clathrin-mediated endocytosis can be triggered, so that we can make it happen on-demand.

Endocytosis on-demand

Using a chemical which diffuses into the cell, we can trigger endocytosis to happen all over the cell. The movie on the right shows vesicles (bright white spots) forming after we add the chemical (at 0:00). The way that we designed the system means that the vesicles that form have one type of cargo in there. This is exciting because it means that we can now deliver things into cells using this cargo. So, we can trigger endocytosis on-demand and we can control the cargo, but we still cannot control where on the plasma membrane this happens.

We solved this problem by engineering a light-sensitive version of our system. With this new version we can use blue light to trigger endocytosis. Whereas the chemical diffused everywhere, the light can be focussed in a narrow region on the cell and endocytosis can be trigger only in that region. This means we control where, as well as when, a vesicle will form.

What does hot-wiring mean?

It is possible to start a car without a key by “hot-wiring” it. This happens in the movies, when the bad guy breaks into a car and just twists some wires together to start the car and make a getaway. To trigger endocytosis we used the cell’s own proteins, but we modified them. We chopped out all the unnecessary parts and just left the bare essentials. We call the process of triggering endocytosis “hot-wiring” because it is similar to just twisting the wires together rather than having a key.

It turns out that movies are not like real life, and hot-wiring a car is actually quite difficult and takes a while. So our systems are more like the Hollywood version than real life!

What is this useful for?

As mentioned above, the systems we have made are useful for cell biologists because they allow cells to be “tamed”. This means that we can accurately study the timing of endocytosis and which proteins are required in a very controlled way. It also potentially means that molecules can be delivered to cells that cannot normally enter. So we have a way to “force feed” cells with whatever we want. This would be most useful for drugs or nanoparticles that are not actively taken up by cells.

Who did the work?

Almost all of the work in the paper was by Laura Wood, a PhD student in the lab. She had help from fellow lab members Nick Clarke, who did the correlative light-electron microscopy, and Sourav Sarkar who did the binding experiments. Gabrielle Larocque, another PhD student did some fantastic work to revise the paper after Laura had departed for a post-doc position at another University. We put the paper up on bioRxiv in Summer 2016 and the paper has slowly made its way through peer review to be published in J Cell Biol today.

Wait? I’m a cell biologist! I want to know how this thing really works!

OK. The design is shown to the right. We made a plasma membrane “anchor” and a clathrin “hook” which is a fragment of protein which binds clathrin. The anchor and the hook have an FRB domain and an FKBP domain and these can be brought together by rapamycin. When the clathrin hook is at the membrane this is recognised by clathrin and vesicle formation can begin. The main hook we use is the appendage and hinge from the beta2 subunit of the AP2 complex.

Normally AP2, which has four subunits, needs to bind to PIP2 in the plasma membrane and undergo a conformational change to recognise a cargo molecule with a specific motif, only then can clathrin bind the beta2 appendage and hinge. By hot-wiring, we effectively remove all of those other proteins and all of those steps to just bring the clathrin binding bit to the membrane when we want. Being able to recreate endocytosis using such a minimalist system was a surprise. In vitro work from Dannhauser and Ungewickell had suggested this might be possible, but it really seems that the steps before clathrin engagement are not a precursor for endocytosis.

To make the light inducible version we used TULIPs (tunable light-controlled interacting proteins). So instead of FRB and FKBP we had a LOVpep and PDZ domain on the hook and anchor.

The post title comes from “Start Me Up” by The Rolling Stones. Originally on Tattoo You, but perhaps better known for its use by Microsoft in their Windows 95 advertising campaign. I’ve finally broken a rule that I wouldn’t use mainstream song titles for posts on this blog.

My Blank Pages V: Raw Data

Raw Data: A novel on Life in Science by Pernille Rørth (Springer, 2016)

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I was keen to read this “lab lit” novel written by renowned cell biologist Pernille Rørth. I’d seen lots of enthusiastic comments about the book, and it didn’t disappoint.

I was frustrated to read two pieces about Raw Data on Retraction Watch and The Node, both of which gave the plot away with no warning, so if you haven’t read it and want to enjoy the suspense while you read, look away now.

The story is set in a high flying cancer cell biology lab in Boston. Postdocs are working night and day to try and land a paper in Nature and to become an independent PI. Chloe manages it, while Karen is struggling, despite her best efforts. Karen accidentally uncovers that Chloe may have cut some corners to get her paper into Nature and this sets off a cascade of events, leading to the retraction of the paper. It’s a fascinating tale, well-written and completely absorbing. I recommend it for anyone working in science. You will smile at the references to conference coffee, failed scientists and more.

The plotline is highly reminiscent of Intuition by Allegra Goodman, even down to the tumours growing in the mice. Both stories echo the real-life events of Thereza Imanishi-Kari which are detailed in the overlong but comprehensive The Baltimore Case by Daniel J. Kevles. Rørth’s retelling of the science world is more convincing that Goodman’s, due in part to her 25 years as a scientist. Nonetheless, Intuition is a great book that I’d also recommend in this genre.

Raw Data is thought-provoking. You can ponder the role of Tom, the PI who has cultivated a certain atmosphere in the lab. What about the pressure to publish? How about the peer reviewers who dangle the carrot of “get this result and you can have a Nature paper” in front of Chloe? It’s a toxic mix and it’s happening in labs all over the world. A terrifying thought.

On the role of Tom: one thing that is slightly underexplored is the fact that Tom tells Chloe that there is a competing group that could ‘scoop’ her while she is rushing to finish her paper. It isn’t clear whether this group actually exists and if this was a tactic to gee her along. Either way it is another bit of pressure which goes on to create the misconduct.

Not so long ago a high profile research institute in the UK announced that it was recruiting PIs by looking for the “best scientific athlete”. I read last week that so far from the London 2012 Olympics, 37 track-and-field sportspeople have had their results disqualified by the IAAF for doping. The parallels are interesting. Science, like sport, is run with winner-takes-all rules and the high stakes and pressure that go along with it. The incentives are dangerous and I wonder what we are creating with this atmosphere. Certainly, as PIs we have a real responsibility, just as coaches do in sport, to ensure our trainees make the right choices in their career.

I’ve seen nothing but recommendations for this book so far and mine is another one.

Here’s Matthew Freeman saying that it would be required reading for everyone in his lab:

My Blank Pages is a track by Velvet Crush. This is an occasional series of book reviews.

My Blank Pages IV: Every Song Ever

Every Song Ever: Twenty Ways to Listen in an Age of Musical Plenty

Ben Ratliff (Farrar, Straus and Giroux)

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A non-science book review for today’s post. This is a great read on “how to listen to music”. There have been hundreds of books published along these lines, the innovation here however is that we now live in an age of musical plenty. Every song ever recorded is available at our fingertips to listen to when, where and how we want. This means that the author can draw on Thelonious Monk, Sunn O))), Shostakovitch and Mariah Carey. And you can seek it out and find out whatever it is that they have in common.

I got hooked in Chapter 2 (discussing slowness in music). I was reading  and thinking: he should mention Sleep’s Dopesmoker, but what are the chances? I turn the page and there it was. Then I knew that we were literally on the same page and that I would enjoy whatever it was he had to say. Isn’t confirmation bias a wonderful  thing (outside of science).

A lot of writing about music is terrible, but I love it when it is done well. As it is here. I especially like reading “under the bonnet” analysis of songs. Ian MacDonald’s Revolution In The Head (or Twilight of the Gods by Wilfred Mellers as an extreme example) springs to mind. This close analysis means you can go back and find new treasures in old songs. And this is the essence of the book.

I must admit that I have thought about trying to write similar analyses of songs on quantixed. Aside from the fact that I don’t have time, I was worried it might make me seem like Patrick Bateman discussing the merits of Huey Lewis & The News in American Psycho. It’s something that’s difficult to do well and Ratliff’s analyses here are light touch and spot-on.

The short section on blast beats which mentioned D.R.I. made me smile too. Although there’s a factual error here. Ratliff talks about how singer-drummer-brother combo Kurt and Eric Brecht lock in on Draft Me when they played CBGB’s in 1984. Drummer Eric had left the band at that point to be replaced by Felix Griffin, and it is him, not Eric, duelling with vocalist Kurt. Both on LP Dealing With It and the gig at CBGB’s which was later released as an LP and video. Again it’s a band that I have soft spot for and it was great to see them picked out.

There were a couple of quotes that I found amusing, being a CD collector and something of a completist. Here’s one:

A friend described to me the experience of acquiring a complete CD collection of Mozart, after having had a piece-by-piece relationship with his music for most of his life. It was 175 CDs, or something like that. “I realized,” he said, “that now that I had it all, I never needed to listen to it again.

Along the same lines, I thought this quote was pretty chilling.

We can pretty much wave bye-bye to the completist-music-collector impulse: it had a limited run in the human brain, probably 1930 to 2010. (It still exists in a fitful way, but it doesn’t have a consensual frame: there is no style for it.) It is not only a way of buying, owning, and arranging music-related objects and experiences in one’s life, but also a distinct way of listening.

 

As somebody who is not a fan of streaming and still values physically owning music I know I am out-of-step with the rest of the world. However I think this quote is at odds with what the whole book is trying to achieve. The guy listening to music on his phone speaker on the bus, described in the intro can’t hear and appreciate much of what is described in the book. To hear that squeak of John Bonham’s kick drum pedal on Since I’ve Been Loving You from Led Zeppelin III, you need to be listening in the old-fashioned way, rather than in the noisy and busy way most music is consumed nowadays.

It’s a great read. You can get it here.

My Blank Pages is a track by Velvet Crush. This is an occasional series of book reviews.

A Day In The Life III

This year #paperOTD (or paper of the day for any readers not on Twitter) did not go well for me. I’ve been busy with lots of things and I’m now reviewing more grants than last year because I am doing more committee work. This means I am finding less time to read one paper per day. Nonetheless I will round up the stats for this year. I only managed to read a paper on 59.2% of available days…

The top ten journals that published the papers that I read:

  • 1 Nat Commun
  • 2 J Cell Biol
  • 3 Nature
  • 4= Cell
  • 4= eLife
  • 4= Traffic
  • 7 Science
  • 8= Dev Cell
  • 8= Mol Biol Cell
  • 8= Nat Cell Biol

Nature Communications has published some really nice cell biology this year and I’m not surprised it’s number one. Also, I read more papers in Cell this year compared to last. The papers I read are mainly recent. Around 83% of the papers were published in 2015. Again, a significant fraction (42%) of the papers have statistical errors. Funnily enough there were no preprints in my top ten. I realised that I tend to read these when approving them as an affiliate (thoroughly enough for #paperOTD if they interest me) but I don’t mark them in the database.

I think my favourite paper was this one on methods to move organelles around cells using light, see also this paper for a related method. I think I’ll try again next year to read one paper per day. I’m a bit worried that if I don’t attempt this, I simply won’t read any papers in detail.

lgs

I also resolved to read one book per month in 2015. I managed this in 2014, but fell short in 2015 just like with #paperOTD. The best book from a limited selection was Matthew Cobb’s Life’s Greatest Secret. A tale of the early days of molecular biology, as it happened. I was a bit sceptical that Matthew could bring anything new to this area of scientific history. Having read Eighth Day of Creation, and then some pale imitations, I thought that this had pretty much been covered completely. This book however takes a fresh perspective and it’s worth reading. Matthew has a nice writing style, animating the dusty old main characters with a insightful detail as he goes along. Check it out.

This blog is going well, with readership growing all the time. I have written about this progress previously (here and here). The most popular posts are those on publishing: preprints, impact factors and publication lag times, rather than my science, but that’s OK. There is more to come on lag times in the New Year, stay tuned.

ladidadiI am a fan of year-end lists as you may be able to tell. My album of the year is Battles – La Di Da Di which came out on Warp in September. An honourable mention goes to Air Formation – Were We Ever Here EP which I bought on iTunes since the 250 copies had long gone by the time I discovered it on AC30.

Since I don’t watch TV or go to the cinema, I don’t have a pick of the year for that. When it comes to pro-cycling, of course I have an opinion. My favourite stage race was Critérium du Dauphiné Libere which was won by Chris Froome in a close contest with Tejay van Garderen. The best one-day race was a tough pick between E3 Harelbeke won by Geraint Thomas and Omloop Het Nieuwsblad won by Ian Stannard. Although E3 was a hard man’s race in tough conditions, I have to go for Stannard outfoxing three(!) Etixx Quick Step riders to take the win in Nieuwsblad. I’m a bit annoyed that those three picks all involve Team Sky and British riders…. I won’t bore everyone with my own cycling (and running) exploits in 2015. Just to say, that I’ve been more active this year in any year since 2009.

I shouldn’t need to tell you where the post title comes from. If you haven’t heard Sgt. Pepper’s Lonely Hearts Club Band by The Beatles, you need to rectify this urgently. The greatest album recorded on 4-track equipment, no question. 🙂

 

My Blank Pages III: The Art of Data Science

largeI recently finished reading The Art of Data Science by Roger Peng & Elizabeth Matsui. Roger, together with Jeff Leek, writes the Simply Statistics blog and he works at JHU with Elizabeth.

The aim of the book is to give a guide to data analysis. It is not meant as a comprehensive data analysis “how to”, nor is it a manual for statistics or programming. Instead it is a high-level guide: how to think about data analysis and how to go about doing it. This makes it an interesting read for anyone working with data.

I think anyone who reads the Simply Statistics blog or who has read the piece Roger and Jeff wrote for Science, will be familiar with a lot of the content in here. At the beginning of the book, I didn’t feel like I learned too much. However, I can see that the “converted” are maybe not the target audience here. Towards the end of the book, the authors walk through a few examples of how to analyse some data focussing on the question in mind, how to refine it and then how to start the analysis. This is the most useful aspect of the book in my opinion, to see the approach to data analysis working in practice. The authors sum up the book early on by comparing it to books about songwriting. I admit to rolling my eyes at this comparison (data analysis as an artform…), but actually it is a good analogy. I think many people who work with data know how to do it, in the way that people who write songs know how to do it, although they probably have not had a formal course in the techniques that are being used. Equally reading a guidebook on songwriting will not make you a great songwriter. A book can only get you so far, intuition and invention are required and the same applies to data science.

The book was published via Lean Pub who have an interesting model where you pay a recommended price (or more!) but if you don’t have the money, you can pay less. Also, you can see what fraction goes to the author(s). The books can be updated continually as typos or code updates are fixed. Roger and the Simply Stats people have put out a few books via this publisher. These books on R, programming, statistics and data science all look good and it seems more books are coming soon.

On a personal note: In 2014, I decided to try and read one book per month. I managed it, but in 2015, I am struggling. It is now November and this book is the 7th I’ve read this year. It was published in September but it took me until now to finish it. Too much going on…

My Blank Pages is a track by Velvet Crush. This is an occasional series of book reviews.

My Blank Pages II: Statistics Done Wrong

I have just finished reading this excellent book, Statistics done wrong: a woefully complete guide by Alex Reinhart. I’d recommend it to anyone interested in quantitative biology and particularly to PhD students starting out in biomedical science.

20150524_214742Statistics is a topic that many people find difficult to grasp. I think there are a couple of reasons for this that I’ll go into below. The aim of this book is to comprehensively cover the common mistakes and errors that are continually crop up in data analysis. The author writes in an easy-to-understand style and – this is the important bit – he dispenses with nearly all the equations. The result is an accessible guide on “what not to do” in significance testing.

I think there are two main reasons why people find statistics tough: uncertainty and mathematical anxiety.

First, uncertainty. What I mean is the uncertainty over what statistical approach to take, rather than the uncertainty that can be studied using statistics! It is very easy to find fault in which statistical approaches have been used in a study by a biologist. Why did they show the confidence interval and not the standard deviation? Why haven’t they corrected for multiple testing…? Statistics has a “gotcha” reputation. The reason for the uncertainty is that it is difficult to come up with a hard-and-fast set of guidelines of approaches to take, because this depends a lot on the type of data that has been collected, what is being tested etc. And there are often several ways to do the same thing. This uncertainty doesn’t go away even with a firm grounding in statistics. The methods are nearly always up for debate as far as I can see. And I think it is this uncertainty that prevents people from really engaging with statistics. In the absence of clear direction, it seems like having in mind a set of “what not to do”, is a useful approach to stats.

Second, mathematical anxiety, i.e. fear of maths. Biology has a reputation for being populated by people who ended up here through an affinity with science but a discomfort with physics and maths. This is unfair as there are many areas of biology where this is not true and statistical/quantitative approaches are right at the forefront. Nonetheless, there is a reason why there are umpteen “Statistics for Biologists” books in the bookshop. Now, the way that statistics is taught is to crunch through the equations that describe statistical concepts. Again, this means that people who really need to know about statistics for their research are held back if they don’t have a mathematical background or just find maths a bit daunting. The situation is well described by a recent post at Will Kurt’s excellent Count Bayesie blog on the teaching of statistics. His point is: insisting that students know these equations gets in the way of them understanding statistics. Nowadays, calculating something like the standard deviation is trivial using a computer and we are unlikely to need to know the derivation of an equation in order to do our work. We should just skip the equations and explain why.

The nice thing about this book is that the author has collected together all the faux pas that you’re likely to encounter and how to avoid them. This goes some way to addressing uncertainty in what methods to use. Secondly, the author has dispensed with the equations, so the mathematically anxious can pick it up without fear. These features make this book different to other stats books that I’ve read.

You can find copies at many online retailers. It’s published by No Starch. I picked up a copy after reading about it on Nathan Yau’s Flowing Data blog.

The post title comes from “My Blank Pages” by Velvet Crush from their Teenage Symphonies to God LP.

Joining A Fanclub

When I started this blog, my plan was to write about interesting papers or at least blog about the ones from my lab. This post is a bit of both.

I was recently asked to write a “Journal Club” piece for Nature Reviews Molecular Cell Biology, which is now available online. It’s paywalled unfortunately. It’s also very short, due to the format. For these reasons, I thought I’d expand a bit on the papers I highlighted.

I picked two papers from Dick McIntosh’s group, published in J Cell Biol in the early 1990s as my subject. The two papers are McDonald et al. 1992 and Mastronarde et al. 1993.

Almost everything we know about the microanatomy of mitotic spindles comes from classical electron microscopy (EM) studies. How many microtubules are there in a kinetochore fibre? How do they contact the kinetochore? These questions have been addressed by EM. McIntosh’s group in Boulder, Colorado have published so many classic papers in this area, but there are many more coming from Conly Rieder, Alexey Khodjakov, Bruce McEwen and many others. Even with the advances in light microscopy which have improved spatial resolution (resulting in a Nobel Prize last year), EM is the only way to see individual microtubules within a complex subcellular structure like the mitotic spindle. The title of the piece, Super-duper resolution imaging of mitotic microtubules, is a bit of a dig at the fact that EM still exceeds the resolution available from super-resolution light microscopy. It’s not the first time that this gag has been used, but I thought it suited the piece quite well.

There are several reasons to highlight these papers over other electron microscopy studies of mitotic spindles.

It was the first time that 3D models of microtubules in mitotic spindles were built from electron micrographs of serial sections. This allowed spatial statistical methods to be applied to understand microtubule spacing and clustering. The software that was developed by David Mastronarde to do this was later packaged into IMOD. This is a great software suite that is actively maintained, free to download and is essential for doing electron microscopy. Taking on the same analysis today would be a lot faster, but still somewhat limited by cutting sections and imaging to get the resolution required to trace individual microtubules.

kfibreThe paper actually showed that some of the microtubules in kinetochore fibres travel all the way from the pole to the kinetochore, and that interpolar microtubules invade the bundle occasionally. This was an open question at the time and was really only definitively answered thanks to the ability to digitise and trace individual microtubules using computational methods.

The final thing I like about these papers is that it’s possible to reproduce the analysis. The methods sections are wonderfully detailed and of course the software is available to do similar work. This is in contrast to most papers nowadays, where it is difficult to understand how the work has been done in the first place, let alone to try and reproduce it in your own lab.

David Mastronarde and Dick McIntosh kindly commented on the piece that I wrote and also Faye Nixon in my lab made some helpful suggestions. There’s no acknowledgement section, so I’ll thank them all here.

References

McDonald, K. L., O’Toole, E. T., Mastronarde, D. N. & McIntosh, J. R. (1992) Kinetochore microtubules in PTK cells. J. Cell Biol. 118, 369—383

Mastronarde, D. N., McDonald, K. L., Ding, R. & McIntosh, J. R. (1993) Interpolar spindle microtubules in PTK cells. J. Cell Biol. 123, 1475—1489

Royle, S.J. (2015) Super-duper resolution imaging of mitotic microtubules. Nat. Rev. Mol. Cell. Biol. doi:10.1038/nrm3937 Published online 05 January 2015

The post title is taken from “Joining a Fanclub” by Jellyfish from their classic second and final LP “Spilt Milk”.

A Day In The Life II

I have been doing paper of the day (#potd) again in 2014. See my previous post about this.

My “rules” for paper of the day are:

  1. Read one paper each working day.
  2. If I am away, or reviewing a paper for a journal or colleague, then I get a pass.
  3. Read it sufficiently to be able to explain it to somebody else, i.e. don’t just scan the abstract and look at the figures. Really read it and understand it. Scan and skim as many other papers as you normally would!
  4. Only papers reporting primary research count. No reviews/opinion pieces etc.
  5. If it was really good or worth telling people about – tweet about it.
  6. Make a simple database in Excel – this helps you keep track, make notes about the paper (to see if you meet #3) and allows you to find the paper easily in the future (this last point turned out to be very useful).

This year has been difficult, especially sticking to #3. My stats for 2014 are:

  • 73% success rate. Down from 85% in 2013
  • Stats errors in 36% of papers I read!
  • 86% of papers were from 2014

Following last year, I wasn’t so surprised by the journals that the papers appeared in:

  1. eLife
  2. J Cell Biol
  3. Mol Biol Cell
  4. Dev Cell
  5. Nature Methods
  6. J Cell Sci
  7. J Neurosci
  8. Nature Cell Biol
  9. Traffic
  10. Curr Biol
  11. Nature
  12. Nature Comm
  13. Science

According to my database I only read one paper in Cell this year. I certainly have lots of them in “Saved for later” in Feedly (which is a black hole from which papers rarely emerge to be read). It’s possible that the reason Cell, Nature and Science are low on the list is that I might quickly glance at papers in those journals but not actually read them for #potd. Last year eLife was at number 9 and this year it is at number 1. This journal is definitely publishing a lot of exciting cell biology and also the lens format is very nice for reading.

I think I’ll try to continue this in 2015. The main thing it has made me realise is how few papers I read (I mean really read). I wonder if students and postdocs are actually the main consumers of the literature. If this is correct, do PIs rely on “subsistence reading”, i.e. when they write their own papers and check the immediate literature? Is their deep reading done only during peer reviewing other people’s work? Or do PIs rely on a constant infusion of the latest science at seminars and at meetings?

Falling and Landing

A great quote from a classic paper by J.B.S. Haldane “On Being The Right Size” (1926).

You can drop a mouse down a thousand-yard mine shaft; and, on arriving at the bottom, it gets a slight shock and walks away, provided that the ground is fairly soft. A rat is killed, a man is broken, a horse splashes.

The paper is available here.


The post title is taken from ‘Falling and Landing’ by The Delgados from their LP ‘Domestiques’.