Background

Endocytosis is the process cells use to internalize macromolecules by forming transport vesicles from the plasma membrane. This process is crucial to many aspects of cell biology because it controls many core functions including cell excitability, cell signalling, and more. The main transport vesicle formed during endocytosis is the clathrin-coated vesicle (CCV). The basic anatomy of a CCV is a cargo-laden vesicle inside a coat. The coat is composed of an outer layer of clathrin, arranged in a beautiful geodesic array of hexagons and pentagons, and an inner layer of adaptors and other proteins that allow clathrin to indirectly contact the cargo and the vesicle.

The quest to define the mechanism of CCV formation using structural methods has a long history. Back in 1969, Kanaseki and Kadota used electron microscopy to describe each CCV as a “vesicle in a basket”¹ and since this time, researchers have followed two paths. In the first, atomic resolution structures of CCV proteins have been obtained predominately using X-ray crystallography. These structures, of individual domains, whole proteins and even some multi-protein complexes have provided valuable snapshots of how the endocytic machinery may go about its job. In the second, much larger assemblies, such as the clathrin cage itself, have been viewed at progressively higher resolution using electron microscopy. What has been missing, until now, was a convergence of these two paths.

What does the assembled clathrin coat on a vesicle look like? Two new preprints use cryo-electron microscopy (cryoEM) to understand CCV assembly. They each use complementary samples and approaches to provide new insight into this fascinating question.

 

 

Key findings

Kovtun et al. used a synthetic strategy to assemble clathrin coats on membranes using purified components². They took a membrane which contained PtdIns(4,5)P₂ and a cargo-like molecule, added a form of AP2 (the main plasma membrane adaptor) and, of course, clathrin. This protein concoction caused clathrin-coated buds to form on the membrane and these invaginations could then be studied by cryoEM, using tomography and subtomogram averaging. Over the last two decades, David Owen’s group have used crystallography to study the AP2 complex in various conformations. Now, together with John Briggs’ tomographic analyses, a view of AP2 posed at the membrane in complex with cargo and relative to clathrin is revealed. We can see for the first time how AP2 interacts with the membrane via multiple PtdIns(4,5)P₂ interactions, and adopts an open conformation which is compatible with cargo binding. This is all happening under the outer layer of clathrin. While the clathrin cage has a regular structure, the underlying AP2 complexes are not organised in a regular lattice-like fashion.

Paraan et al. started with clathrin-coated vesicles isolated from bovine brains³. Using cryoEM and single particle analysis we see high-resolution views of the clathrin coat. The power of this approach is in the use of a native sample. Scott Stagg’s group found a range of clathrin cage types, which echoed recent work on clathrin cages assembled in vitro by Corinne Smith’s lab⁴. In fact, they observed a new type of clathrin geometry, dubbed the C2 cage, which appears to break the head-to-tail exclusion model for cage assembly; a reminder that biology is a messy business where rule-breaking is the norm. Because this is a native sample, AP2 and a number of other proteins are present in the CCVs. This means that there is some uncertainty in assigning densities to certain molecules. Nevertheless, density which matches the size and shape of AP2 could be resolved in the inner layer of the coat.

AP2 has two appendage domains on the alpha and beta2 subunits which attach to the core heterotetramer via long, flexible hinges. We know that beta2 engages clathrin, but what these studies now reveal is how this happens in an assembled coat. Both studies find the beta2 appendage to be present in the hexagonal faces rather than the pentagonal ones. Since an enclosed cage must contain twelve pentagons, the creation of pentagons is key to driving vesicle curvature. The preference of beta2 appendage for hexagonal versus pentagonal faces may explain why AP2 drives vesicle curvature via clathrin, even though it has no curvature-generating properties by itself.

There is a wealth of further detail in these studies that will satisfy the clathrin aficionados (like me!). For example, the beta2 appendage position relative to the clathrin legs is different in each study. Again, highlighting that any “rules” for clathrin engagement by AP2 may be easily broken.

 

What I liked about these preprints

Both preprints represent a significant step forward in our understanding of CCV formation. The work underlying this advance is very technically challenging. Clathrin assemblies are large, flexible and heterogeneous. All three properties make structural approaches – which rely on averaging – much more difficult. The complementarity of the two studies is nice: Paraan et al. used native material and single particle methods, while Kovtun et al. went for a reductionist approach and tomographic imaging.

CryoEM has revolutionised structural biology. We are now at an exciting point where these high-resolution views of complex samples are not only possible, but also have the power to reveal so much, thanks to years of previous work in crystallography, cell biology and biochemistry. These preprints are perfect examples of the point that we’ve reached.

 

Acknowledgements

I would like to thank Sarah Smith and Katie Wood from Corinne Smith’s lab for valuable discussions around these preprints.

 

References

1. Kanaseki & Kadota (1969) The “vesicle in a basket”. A morphological study of the coated vesicle isolated from the nerve endings of the guinea pig brain, with special reference to the mechanism of membrane movements. J Cell Biol, 42, 202-20.
2. Kovtun et al. (2020) Architecture of the AP2:clathrin coat on the membranes of clathrin-coated vesicles. bioRxiv, 2020.01.28.922591.
3. Paraan et al. (2020) The Structures of Natively Assembled Clathrin Coated Vesicles. bioRxiv, 2020.01.28.923128.
4. Morris et al. (2019) Cryo-EM of multiple cage architectures reveals a universal mode of clathrin self-assembly. Nat Struct Mol Biol, 26, 890-898.

Tags: ap2, clathrin, cryoem, endocytosis

doi: https://doi.org/10.1242/prelights.17130

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