Vesicles: Looking inside the cell

Advances in imaging techniques have shed new light on the structure of vesicles formed by COPI protein complexes.
  1. Eric C Arakel
  2. Blanche Schwappach  Is a corresponding author
  1. University Medical Center Göttingen, Germany

Vesicles perform a wide range of functions within cells, such as the transport of proteins and lipids between the different parts of a cell. Each vesicle is coated with a protein complex, and understanding the structure and function of these complexes is a central challenge in cell biology. Two of these complexes – COPII and clathrin – are relatively well understood, but the third, which is called COPI, is not.

Vesicles coated with COPI are responsible for transport from the Golgi to the endoplasmic reticulum, and also for transport between the cisternae within the Golgi (Beck et al., 2009). The COPI complex contains seven subunits that are recruited en bloc to the vesicle membrane (Hara-Kuge et al., 1994). However, the intricate couplings between these subunits have long hindered efforts to determine the molecular architecture of the COPI coats.

The basic model of COPI vesicle formation begins with the recruitment of a GTPase called Arf1 to a membrane. Arf1 is then loaded with GTP, thereby activating it. COPI complexes then interact with the GTP-loaded Arf1 and cargo proteins to form the vesicle, which can then detach from the membrane and transport the cargo proteins to their destination. Finally, the coat disassembles in a process that is called un-coating: three GTPase activating proteins (ArfGAP1/2/3) play an important role in this process by stimulating the hydrolysis of the GTP and, therefore, deactivating Arf1 (Lanoix et al., 1999; Pepperkok et al., 2000; Weimer et al., 2008). Subsequent studies of this un-coating process suggested that Arf1 and COPI disengage from membranes independently, in a non-cooperative manner (Presley et al., 2002; Yang et al., 2002). These and other results led to a model in which the individual COPI complexes were held in place by lateral linkages within the coat and by interactions with the underlying cargo.

These studies were built upon in a series of in-vitro reconstitution experiments undertaken by a collaboration between the groups of Felix Wieland (University of Heidelberg) and John Briggs (European Molecular Biology Laboratory and the MRC Laboratory of Molecular Biology). The in-vitro reconstitution approach involves combining purified proteins and liposomes (a type of vesicle) in a test tube to generate COPI-coated vesicles, thus allowing researchers to study the precise sequence of events that take place during vesicle formation. Wieland, Briggs and co-workers exploited significant advances in two techniques – cryo-electron tomography and subtomogram averaging – to determine the molecular structure of the COPI coat (Dodonova et al., 2017; Dodonova et al., 2015; Faini et al., 2012).

However, the process of vesicle formation in these seminal experiments differed from the process in real cells in a number of ways: the vesicles did not contain any cargo, and the Arf1 was frozen in its activated state because the researchers used a variant of GTP called GTPγS that cannot be hydrolysed. This meant that several questions remained unanswered: Does early GTP hydrolysis contribute to a pronounced conformational change of the coat (such as the exposure of cargo-recognition sites)? Are the linkages within the coat rearranged to accommodate cargo? How does Arf1 disengage from COPI without perturbing the pre-existing lattice? And how does the cage adapt structurally to the loss of Arf1?

The rational next step would have involved using GTP (rather than GTPγS), the three ArfGAPs and cargo proteins in in-vitro reconstitution experiments, which would have been very arduous. However, in an unexpected development reported in eLife, Briggs, Benjamin Engel and Wolfgang Baumeister (both of the Max Planck Institute of Biochemistry) and co-workers – including Yury Bykov as first author – describe the native structure of COPI-coated vesicles in a species of green algae called Chlamydomonas reinhardtii (Bykov et al., 2017). This quantum leap was made possible through a combination of cryo-focused ion-beam milling (a technique that involves vitrifying cells and then using a focused ion beam to slice off ultrathin sections) and further improvements in cryo-electron tomography and subtomogram averaging. Moreover, this approach meant that the researchers were able to study the structure of the coat in the presence of active Arf1, the relevant ArfGAPs and the vesicle's cargo.

Building on their previous systematic characterization of the morphology of the Golgi in C. reinhardtii (Engel et al., 2015a; Engel et al., 2015b), and what is known about the structures of COPI, COPII and clathrin, Bykov et al. were able to identify the three different types of coated vesicles in the vicinity of the cisternae within the Golgi. The researchers were also able to determine the in-situ structure of the coat without relying on existing structural models, and found that it was remarkably similar to the structure that emerged from the in-vitro reconstitution experiments. This finding is significant for the field because it strongly suggests that the existing structural model is physiologically relevant.

Moreover, an additional density was observed inside the vesicles in C. reinhardtii that was not visible in the in-vitro experiments. This density is likely to correspond to cargo, and the fact that it was observed under the β-propeller of the β’-subunit, but not beneath the β-propeller of the α-subunit (which is structurally similar), suggests that this cargo is recognized by a specific subunit of COPI. It is also striking that this density is seen in many vesicles adjoining both the cis and medial cisternae.

Remarkably, the relative proportions of Arf1 and COPI were the same for most vesicles, irrespective of the intactness of the coat: this suggests that, upon hydrolysis, the constituents of the coat are all released at the same time to allow the vesicle to fuse with an acceptor compartment, and argues against a step-wise dissociation of the coat from the vesicle.

In addition to demonstrating the ability of in-situ experiments to distinguish between models based on in-vitro experiments, the work of Bykov et al. highlights the ability of new techniques to allow us to see things – such as the density under the β-propeller of the β’-subunit – that we have missed so far. We are keen to get a glimpse of COPII and clathrin in situ too.

References

    1. Hara-Kuge S
    2. Kuge O
    3. Orci L
    4. Amherdt M
    5. Ravazzola M
    6. Wieland FT
    7. Rothman JE
    (1994)
    En bloc incorporation of coatomer subunits during the assembly of COP-coated vesicles
    Journal of Cell Biology 124:883–892.
    1. Pepperkok R
    2. Whitney JA
    3. Gomez M
    4. Kreis TE
    (2000)
    COPI vesicles accumulating in the presence of a GTP restricted arf1 mutant are depleted of anterograde and retrograde cargo
    Journal of Cell Science 113 (Pt 1):135–144.

Article and author information

Author details

  1. Eric C Arakel

    Eric C Arakel is in the Department of Molecular Biology, University Medical Center Göttingen, Göttingen, Germany

    Competing interests
    No competing interests declared
  2. Blanche Schwappach

    Blanche Schwappach is in the Department of Molecular Biology, University Medical Center Göttingen, Göttingen, Germany

    For correspondence
    blanche.schwappach@med.uni-goettingen.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0225-6432

Publication history

  1. Version of Record published: December 5, 2017 (version 1)

Copyright

© 2017, Arakel et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,035
    Page views
  • 255
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Eric C Arakel
  2. Blanche Schwappach
(2017)
Vesicles: Looking inside the cell
eLife 6:e33650.
https://doi.org/10.7554/eLife.33650

Further reading

    1. Structural Biology and Molecular Biophysics
    Josep Rizo et al.
    Research Article Updated

    Synaptic vesicles are primed into a state that is ready for fast neurotransmitter release upon Ca2+-binding to Synaptotagmin-1. This state likely includes trans-SNARE complexes between the vesicle and plasma membranes that are bound to Synaptotagmin-1 and complexins. However, the nature of this state and the steps leading to membrane fusion are unclear, in part because of the difficulty of studying this dynamic process experimentally. To shed light into these questions, we performed all-atom molecular dynamics simulations of systems containing trans-SNARE complexes between two flat bilayers or a vesicle and a flat bilayer with or without fragments of Synaptotagmin-1 and/or complexin-1. Our results need to be interpreted with caution because of the limited simulation times and the absence of key components, but suggest mechanistic features that may control release and help visualize potential states of the primed Synaptotagmin-1-SNARE-complexin-1 complex. The simulations suggest that SNAREs alone induce formation of extended membrane-membrane contact interfaces that may fuse slowly, and that the primed state contains macromolecular assemblies of trans-SNARE complexes bound to the Synaptotagmin-1 C2B domain and complexin-1 in a spring-loaded configuration that prevents premature membrane merger and formation of extended interfaces, but keeps the system ready for fast fusion upon Ca2+ influx.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Tsuyoshi Imasaki et al.
    Research Article

    Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αβ-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.