Not What You Want: our new paper on a side effect of GFP nanobodies

We have a new preprint out – it is a cautionary tale about using GFP nanobodies in cells. This short post gives a bit of background to the work. Please read the paper if you are interested in using GFP nanobodies in cells, you can find it here.

Paper in a nutshell: Caution is needed when using GFP nanobodies because they can inhibit their target protein in cells.

People who did the work: Cansu Küey did most of the work for the paper. She discovered the inhibition side effect of the dongles. Gabrielle Larocque contributed a figure where she compared dongle-knocksideways with regular knocksideways. The project was initiated by Nick Clarke who made our first set of dongles and tested which fluorescent proteins the nanobody binds in cells. Lab people and their profiles can be found here.

Background: Many other labs have shown that nanobodies can be functionalised so that you can stick new protein domains onto GFP-tagged proteins to do new things. This is useful because it means you can “retrofit” an existing GFP knock-in cell line or organism to do new things like knocksideways without making new lines. In fact there was a recent preprint which described a suite of functionalised nanobodies that can confer all kinds of functions to GFP.

Like many other labs we were working on this method. We thought functionalised GFP nanobodies resembled “dongles” – those adaptors that Apple makes so much money from – that convert one port to another.

Dongles, dongles, dongles… (photo by Rex Hammock, licensed for reuse https://www.flickr.com/photos/rexblog/5575298582)

A while back we made several different dongles. We were most interested in a GFP nanobodies with an additional FKBP domain that would allow us to do knocksideways (or FerriTagging) in GFP knock-in cells. For those that don’t know, knocksideways is not a knockout or a knockdown, but a way of putting a protein somewhere else in the cell to inactivate it. The most common place to send a protein is to the mitochondria.

Knocksideways works by joining FKBP and FRB (on the mitochondria) using rapamycin. Normally FKBP is fused to the protein of interest (top). If we just have a GFP tag, we can’t do knocksideways (middle). If we add a dongle (bottom) we can attach FKBP domains to allow knocksideways to happen.

We found that dongle-knocksideways works really well and we were very excited about this method. Here we are removing GFP-clathrin from the mitotic spindle in seconds using dongle knocksideways.

GFP-clathrin, shown here in blue is sent to the mitochondria (yellow) using rapamycin. This effect is only possible because of the dongle which adds FKBP to GFP via a GFP nanobody.

Since there are no specific inhibitors of endocytosis, we thought dongle knocksideways would be cool to try in cells with dynamin-2 tagged with GFP at both alleles. There is a line from David Drubin’s lab which is widely used. This would mean we could put the dongle plasmids on Addgene and everyone could inhibit endocytosis on-demand!

Initial results were encouraging. We could put dynamin onto mitochondria alright.

Dynamin-2-GFP undergoing dongle-knocksideways. The Mitotrap is shown in red and dynamin is green.

But we hit a problem. It turns out that dongle binding to dynamin inhibits endocytosis. So we have unintended inhibition of the target protein. This is a big problem because the power of knocksideways comes from being able to observe normal function and then rapidly switch it off. So if there is inhibition before knocksideways, the method is useless.

Now, this problem could be specific to dynamin or it might be a general problem with all targets of dongles. Either way, we’ve switched from this method and wrote this manuscript to alert others to the side effects of dongles. We discuss possible ways forward for this method and also point out some applications of the nanobody technology that are unaffected by our observations.

The post title comes from “Not What You Want” by Sleater-Kinney from their wonderful Dig Me Out record.

All That Noise: The vesicle packing problem

This week Erick Martins Ratamero and I put up a preprint on vesicle packing. This post is a bit of backstory but please take a look at the paper, it’s very short and simple.

The paper started when I wanted to know how many receptors could fit in a clathrin-coated vesicle. Sounds like a simple problem – but it’s actually more complicated.

Of course, this problem is not as simple as calculating the surface area of the vesicle, the cross-sectional area of the receptor and dividing one by the other. The images above show the problem. The receptors would be the dimples on the golf ball… they can’t overlap… how many can you fit on the ball?

It turns out that a PhD student working in Groningen in 1930 posed a similar problem (known as the Tammes Problem) in his thesis. His concern was the even pattern of pores on a pollen grain, but the root of the problem is the Thomson Problem. This is the minimisation of energy that occurs when charged particles are on a spherical surface. The particles must distribute themselves as far away as possible from all other particles.

There are very few analytical solutions to the Tammes Problem (presently 3-14 and 24 are solved). Anyhow, our vesicle packing problem is the other way around. We want to know, for a vesicle of a certain size, and cargo of a certain size, how many can we fit in.

Fortunately stochastic Tammes solvers are available like this one, that we could adapt. It turns out that the numbers of receptors that could be packed is enormous: for a typical clathrin-coated vesicle almost 800 G Protein-Coupled Receptors could fit on the surface. Note, that this doesn’t take into account steric hinderance and assumes that the vesicle carries nothing else. Full details are in the paper.

Why does this matter? Many labs are developing ways to count molecules in cellular structures by light or electron microscopy. We wanted to have a way to check that our results were physically possible. For example, if we measure 1000 GPCRs in a clathrin-coated vesicle, we know something has gone wrong.

What else? This paper ticked a few things on my publishing bucket list: a paper that is solely theoretical, a coffee-break idea paper and one that is on a “fun” subject. Erick has previous form with theoretical/fun papers, previously publishing on modelling peloton dynamics in procycling.

We figured the paper was more substantial than a blog post yet too minimal to send to a journal. So unless a journal wants to publish it (and gets in touch with us), this will be my first preprint where bioRxiv is the final destination.

We got a sense that people might be interested in an answer to the vesicle packing problem because whenever we asked people for an estimate, we got hugely different answers! The paper has been well-received so far. We’ve had quite a few comments on Twitter and we’re glad that we wrote up the work.

The post title comes from the “All That Noise” LP by The Darkside. I picked this not because of the title, but because of the cover.

All That Noise cover shows a packing problem on a sphere

Voice Your Opinion: Editors shopping for preprints is the future

Today I saw a tweet from Manuel Théry (an Associate Ed at Mol Biol Cell). Which said that he heard that the Editor-in-Chief of MBoC, David Drubin shops for interesting preprints on bioRxiv to encourage the authors to submit to MBoC. This is not a surprise to me. I’ve read that authors of preprints on bioRxiv have been approached by journal Editors previously (here and here, there are many more). I’m pleased that David is forward-thinking and that MBoC are doing this actively.

I think this is the future.

Why? If we ignore for a moment the “far future” which may involve the destruction of most journals, leaving a preprint server and a handful of subject-specific websites which hunt down and feature content from the server and co-ordinate discussions and overviews of current trends… I actually think this is a good idea for the “immediate future” of science and science publishing. Two reasons spring to mind.

  1. Journals would be crazy to miss out: The manuscripts that I am seeing on bioRxiv are not stuff that’s been dumped there with no chance of “real publication”. This stuff is high profile. I mean that in the following sense: the work in my field that has been posted is generally interesting, it is from labs that do great science, and it is as good as work in any journal (obviously). For some reason I have found myself following what is being deposited here more closely than at any real journal. Journals would be crazy to miss out on this stuff.
  2. Levelling the playing field: For better-or-worse papers are judged on where they are published. The thing that bothers me most about this is that manuscripts are only submitted to 1 or more journals before “finding their home”. This process is highly noisy and it means that if we accept that there is a journal hierarchy, your paper may or may not be deserving of the kudos it receives in its resting place. If all journals actively scour the preprint server(s), the authors can then pick the “highest bidder”. This would make things fairer in the sense that all journals in the hierarchy had a chance to consider the paper and its resting place may actually reflect its true quality.

I don’t often post opinions here, but I thought this would take more than 140 characters to explain. If you agree or disagree, feel free to leave a comment!

Edit @ 11:46 16-05-26 Pedro Beltrao pointed out that this idea is not new, a post of his from 2007.

Edit 16-05-26 Misattributed the track to Extreme Noise Terror (corrected). Also added some links thanks to Alexis Verger.

The post title comes from “Voice Your Opinion” by Unseen Terror. The version I have is from a Peel sessions compilation “Hardcore Holocaust”.

White label: the growth of bioRxiv

bioRxiv, the preprint server for biology, recently turned 2 years old. This seems a good point to take a look at how bioRxiv has developed over this time and to discuss any concerns sceptical people may have about using the service.

Firstly, thanks to Richard Sever (@cshperspectives) for posting the data below. The first plot shows the number of new preprints deposited and the number that were revised, per month since bioRxiv opened in Nov 2013. There are now about 200 preprints being deposited per month and this number will continue to increase. The cumulative article count (of new preprints) shows that, as of the end of last month, there are >2500 preprints deposited at bioRxiv. overall2

subject2

What is take up like across biology? To look at this, the number of articles in different subject categories can be totted up. Evolutionary Biology, Bioinformatics and Genomics/Genetics are the front-running disciplines. Obviously counting articles should be corrected for the size of these fields, but it’s clear that some large disciplines have not adopted preprinting in the same way. Cell biology, my own field, has some catching up to do. It’s likely that this reflects cultures within different fields. For example, genomics has a rich history of data deposition, sharing and openness. Other fields, less so…

So what are we waiting for?

I’d recommend that people wondering about preprinting go and read Stephen Curry’s post “just do it“. Any people who remain sceptical should keep reading…

Do I really want to deposit my best work on bioRxiv?

I’ve picked six preprints that were deposited in 2015. This selection demonstrates how important work is appearing first at bioRxiv and is being downloaded thousands of times before the papers appear in the pages of scientific journals.

  1. Accelerating scientific publishing in biology. A preprint about preprinting from Ron Vale, subsequently published in PNAS.
  2. Analysis of protein-coding genetic variation in 60,706 humans. A preprint summarising a huge effort from ExAC Exome Aggregation Consortium. 12,366 views, 4,534 downloads.
  3. TP53 copy number expansion correlates with the evolution of increased body size and an enhanced DNA damage response in elephants. This preprint was all over the news, e.g. Science.
  4. Sampling the conformational space of the catalytic subunit of human γ-secretase. CryoEM is the hottest technique in biology right now. Sjors Scheres’ group have been at the forefront of this revolution. This paper is now out in eLife.
  5. The genome of the tardigrade Hypsibius dujardini. The recent controversy over horizontal gene transfer in Tardigrades was rapidfire thanks to preprinting.
  6. CRISPR with independent transgenes is a safe and robust alternative to autonomous gene drives in basic research. This preprint concerning biosafety of CRISPR/Cas technology could be accessed immediately thanks to preprinting.

But many journals consider preprints to be previous publications!

Wrong. It is true that some journals have yet to change their policy, but the majority – including Nature, Cell and Science – are happy to consider manuscripts that have been preprinted. There are many examples of biology preprints that went on to be published in Nature (ancient genomes) and Science (hotspots in birds). If you are worried about whether the journal you want to submit your work to will allow preprinting, check this page first or the SHERPA/RoMEO resource. The journal “information to authors” page should have a statement about this, but you can always ask the Editor.

I’m going to get scooped

Preprints establish priority. It isn’t possible to be scooped if you deposit a preprint that is time-stamped showing that you were the first. The alternative is to send it to a journal where no record will exist that you submitted it if the paper is rejected, or sometimes even if they end up publishing it (see discussion here). Personally, I feel that the fear of scooping in science is overblown. In fields that are so hot that papers are coming out really fast the fear of scooping is high, everyone sees the work if its on bioRxiv or elsewhere – who was first is clear to all. Think of it this way: depositing a preprint at bioRxiv is just the same as giving a talk at a meeting. Preprints mean that there is a verifiable record available to everyone.

Preprints look ugly, I don’t want people to see my paper like that.

The depositor can format their preprint however they like! Check out Christophe Leterrier’s beautifully formatted preprint, or this one from Dennis Eckmeier. Both authors made their templates available so you can follow their example (1 and 2).

Yes but does -insert name of famous scientist- deposit preprints?

Lots of high profile scientists have already used bioRxiv. David Bartel, Ewan Birney, George Church, Ray Deshaies, Jennifer Doudna, Steve Henikoff, Rudy Jaenisch, Sophien Kamoun, Eric Karsenti, Maria Leptin, Rong Li, Andrew Murray, Pam Silver, Bruce Stillman, Leslie Vosshall and many more. Some sceptical people may find this argument compelling.

I know how publishing works now and I don’t want to disrupt the status quo

It’s paradoxical how science is all about pushing the frontiers, yet when it comes to publishing, scientists are incredibly conservative. Physics and Mathematics have been using preprinting as part of the standard route to publication for decades and so adoption by biology is nothing unusual and actually, we will simply be catching up. One vision for the future of scientific publishing is that we will deposit preprints and then journals will search out the best work from the server to highlight in their pages. The journals that will do this are called “overlay journals”. Sounds crazy? It’s already happening in Mathematics. Terry Tao, a Fields medal-winning mathematician recently deposited a solution to the Erdos discrepency problem on arXiv (he actually put them on his blog first). This was then “published” in Discrete Analysis, an overlay journal. Read about this here.

Disclaimer: other preprint services are available. F1000 Research, PeerJ Preprints and of course arXiv itself has quantitative biology section. My lab have deposited work at bioRxiv (1, 2 and 3) and I am an affiliate for the service, which means I check preprints before they go online.

Edit 14/12/15 07:13 put the scientists in alphabetical order. Added a part about scooping.

The post title comes from the term “white label” which is used for promotional vinyl copies of records ahead of their official release.