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.
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.
Recently, Ron Vale put up this very interesting piece on bioRxiv discussing what it takes to publish a paper in the field of cell biology these days. In the main, he questions whether this is now out of reach of many trainees in our labs. It raises some great points and I recommend reading it.
One (of many) interesting stats in the article is that J Cell Biol now publishes fewer papers than it used to. Which made me think back to the photo and wonder why there has been a decline. Elsewhere, Vale notes that a cell biology paper now contains >2 the amount of data than papers of yesteryear. I’ve also written before about the creeping increase in the number of authors per paper at J Cell Biol and (more so) at Cell. Publication in Science is something of an arms race and his point is really that the amount of data, the time taken, the effort/people involved has got to an untenable level.
The data in the preprint is a bit limited as he only looks at two snapshots in time – because he looks at two cohorts of students at UCSF. So I thought I’d look at the decrease in JCB papers over time – did it really fall off? by how much? when did it start?.
Getting the data is straightforward. In fact, PubMed will give you a csv of frequency of papers for a given search term (it even shows you a snapshot in the main search window). I wanted a bit more control, so I exported the records for JCB and NCB. I filtered out interviews and commentary as best as I could and plotted out the records as two histograms using a bin width of 6 months. It’s pretty clear that J Cell Biol is indeed publishing fewer papers now than it used to. It looks like the trend started around 2002, possibly accelerating in the last 5 years (the photo agrees with this). The six month output at JCB in 2015 is similar to what it was in 1975!
In the comments section of the preprint, there is a bit of discussion of why this may be. Overall, there are more and more papers being published every year. There’s no reason to think that the number of cell biology papers has remained static or fallen. So if J Cell Biol have not taken a decision to limit the number of papers, why is there a decline? One commenter suggests Nature Cell Biology has “taken” some of these papers. So I plotted those numbers out too. The number of papers at NCB is capped and has been constant since the launch of the journal. It does look like NCB could be responsible, but it’s a complex question. Personally, I think it’s unlikely. When NCB was launched this marked a period of expansion in the number of scientific journals and it’s likely that the increase in number of venues that a paper can go to (rather than the creation of NCB per se) has affected publication at JCB. One simple cause could be financial, i.e. the page number being limited by RUP. If this is true, why not move the journal online? There’s so many datasets and movies in papers these days that it barely makes sense to print JCB any more.
I love reading papers in JCB. They are sufficiently detailed so that you know what’s going on. They’re definitely on Cell Biology, not some tangential area of molecular biology. The Editors are active cell biologists and it has had a long history of publishing some truly landmark discoveries in our field. For these reasons, I’m sad that there are fewer JCB papers these days. If it’s an editorial decision to try to make the journal more exclusive, this is even more regrettable. I wonder if the Editors feel that they just don’t get enough high quality papers. If this is the case, then maybe the expectations for what a paper “should be” need to be brought back in line with reality. Which is one of the points that Ron Vale is making in his article.
* I cropped the picture to remove some identifying things on the bookshelf.
Update @ 07:07 17/7/15: Rebecca Alvinia from JCB had left a comment on Ron Vale’s piece on bioRxiv to say that JCB are not purposely limiting the number of papers. Fillip Port then asked why JCB does not take preprints. Rebecca has now replied saying that following a change of policy, J Cell Biol and the other RUP journals will take preprinted papers. This is great news!
Creep Diets is the title track from the second album by the oddly named Fudge Tunnel, released on Earache Records in 1993
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.
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.
The 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.
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”.