Don’t Let Go The Coat: new paper on clathrin assembly

A new paper means a new paper explainer. This post is all about our new paper on clathrin assembly.

Some background info

Endocytosis is the way that cells take up material from the outside world. The cell can make tiny vesicles that bud inwards from the cell surface and pinch off to travel inside the cell. This process is important for lots of things that cells do, so there is a lot of interest in how endocytosis works.

The main type of vesicle formed during endocytosis has a clathrin coat. It’s a polygonal assembly (hexagons and pentagons) of a protein called clathrin. This cage looks a bit like a football. What we looked at in this paper is how the clathrin cage is assembled so quickly during endocytosis.

This is Figure 1 from the paper. Clathrin afficianados may appreciate the retro Musacchio-ised graphics in panel D. The code to do that is described here.

Clathrin, the three-legged “triskelion” shown above, can be purified from tissue. It spontaneously assembles into the football shapes in the test tube. These assemblies can be imaged by electron microscopy and a high resolution view emerges (I wrote a bit about that here).

Clathrin’s secret sauce

The footballs assemble spontaneously, but there are conditions that affect how well assembly works. One secret assembly ingredient is a protein (hinge and appendage of beta2) which accelerates the process tremendously. This has been known for many years. The image below is over twenty years old.

Beta2 hinge and appendage – the secret sauce. Image is from Figure 6 in Owen et al. 2000

This observation is puzzling since it is known that beta2 hinge and appendage (part of the AP2 complex) binds clathrin but it was thought that this is to do with bringing clathrin to the membrane right at the start of endocytosis.

Sarah Smith had assembled clathrin using this special ingredient and realised that by taking EM images of thousands of these assemblies she could find how beta2 binds to clathrin to accelerate its assembly. To her surprise she found it bound in two different places in the cage. One place had previously been reported (more on this below) and another was completely new.

Two really nice papers were published last year showing that beta2 binds in two different locations in the clathrin cage (Kovtun et al., 2020 and Paraan et al., 2020). Each paper used a different method of preparing the cages and had come to different conclusions about where beta2 binds. Sarah’s data brought the total number of modes of binding to three! So it seems that this binding is multi-modal. There may be even more binding sites!

What does this tell us? Well, when looked at each mode of binding we noticed that they all have one thing in common: they crosslink distinct triskelia. This would explain why beta2 is not just needed to bring clathrin initially, but it accelerates assembly by crosslinking the units in the assembly. It also consolidates the findings of all three papers.

Class 15 and 18 were the two modes found by Sarah. The models of Kovtun and Paraan et al are also shown. This is Figure 8 from Smith et al. 2021

We contributed some experiments in live cells. We have a trick where we can measure endocytosis which is induced by beta2 hinge and appendage alone – termed hot-wiring. This was important to translate what Corinne’s group has seen by EM with what happens functionally in the cell during endocytosis.

The people

This work was a collaboration between our lab and Corinne Smith’s group. Gabrielle Larocque led the cell biology side and Sarah Smith led the structural biology (cryoEM) work. Katie Wood helped with cryoEM experiments and Kyle Morris helped with structural analysis. Alan Roseman and Richard Sessions at Manchester and Bristol, respectively, contributed computational analyses.

Publication process

We put the manuscript up on bioRxiv on May 22 2021 and submitted it to EMBO J. We got some very supportive comments from the reviewers and we were able to address these with some small changes. This is the first minor revisions paper I have been involved with for a long time! It was accepted on Aug 3 2021, which makes it one of the quickest papers I’ve been involved with too. We were all very happy with the handling of the paper.

The only bump in the road was a catch 22 we found ourselves in. We had deposited our structural data at EMDB and EMPIAR but had not released it with the preprint. It’s possible to embargo the release until the paper comes out. At the revision stage, the journal asked us to release the data. This meant we somehow needed to release the data and link it to publication but the paper was not yet accepted. It wasn’t clear to us whether the data could be retrospectively attributed to the paper. I’m curious to know what other labs do in this situation. I guess one solution is to just release everything with the preprint, which would have got around this problem.

The paper is open access, which means anyone can read it. It has a CC-BY 4.0 licence. The open access fee was paid by agreement between the commercial publisher Wiley and a consortium of UK University libraries. You can read it here.

The post title comes from “Don’t Let Go The Coat” by The Who. I’m not a fan of the song, but the title was apt and it’s in my library so…