Award details

Understanding mechanisms of coat assembly and membrane deformation.

Reference	BB/T002670/1
Principal Investigator / Supervisor	Dr Giulia Zanetti
Co-Investigators / Co-Supervisors
Institution	Birkbeck College
Department	Biological Sciences
Funding type	Research
Value (£)	525,227
Status	Current
Type	Research Grant
Start date	07/01/2020
End date	06/07/2023
Duration	42 months

Abstract

In membrane trafficking, coat complexes assemble on the bilayer and induce its deformation into vesicles, while also mediating cargo incorporation. One of the fundamental characteristics of coats is their ability to achieve and control flexibility and plasticity of membrane remodelling, yet the molecular basis for this remains a major outstanding question. We aim to address it by working on the structural and functional characterisation of the coat protein complex II (COPII). Our recent work led us to hypothesise that a network of partially redundant interactions promote coat assembly and membrane deformation in an inter-dependent manner, serving as a platform for regulation of membrane remodelling. The project proposed here timely addresses this hypothesis and leads to formulate new avenues to understand COPII regulation. We will specifically tackle three objectives: 1. To determine the role of assembly of the COPII inner and outer coat layers. We will use in vitro reconstitution of COPII budding from GUVs and use cryo-tomography and subtomogram averaging to assess the nature of coat interactions and dissect their contribution to membrane deformation. 2. To determine the role of assembly/disassembly kinetics. We will reconstitute budding in the presence of GTP and assess vesicle/tubule formation by time-resolved EM, concomitantly with biochemical assays to quantify membrane recruitment and GTP hydrolysis rates. 3. To determine the role of cargo. We will reconstitute cargo proteins into GUVs, and analyse membrane and coat architecture by cryo-EM, together with analysis of uncoating kinetics by time-resolved EM. This project will advance our molecular understanding of COPII, opening up new avenues of research into ill-defined regulation mechanisms. Our work will also set the basis and serve as a reference to study many other systems involving membrane remodelling by coats and will therefore contribute to elucidate a broad and fundamental aspect of biology.

Summary

Cells can be likened to cities to describe their structure and organisation. The cell's citizens are the proteins, which carry out all the activities necessary to maintain cell homeostasis and ensure survival and proliferation. Eukaryotic cells are organised in regions delimited by membranes which are called compartments. These can be thought of as precisely defined neighbourhoods where particular activities take place. For example an organelle called the endoplasmic reticulum (shortened to ER) is where certain classes of proteins are made, and where the initial checks on their health are performed. Another important organelle is the Golgi: this is where proteins mature into their fully functional form and are sorted to where they'll perform their job. As in cities, proteins need to be transported from one compartment to another - a fundamental aspect that is necessary to maintain cell functionality. Because cell compartments are delimited by membranes, communication between compartments and exchange of material happens through vesicles: small sacs of membrane bud and pinch off from the originating compartment loaded with cargo, to then release it to the target compartment. The transport link that connects the ER to the Golgi, to get young proteins from where they were born to where they will fully mature is called COPII. COPII is a set of proteins which aid formation of vesicles from the ER by assembling into curved scaffolds around the ER membrane leading it to bud a vesicle (COPII thereby forms a so-called "coat"). While deforming the ER membrane, COPII also links with proteins inside the ER that need to be transported (cargo), so that these are incorporated in the forming vesicle. Deformation of the ER membrane follows the coat shape. We have a good functional understanding of COPII mechanisms, but our view of the coat in action on membranes is somewhat fuzzy. In particular we do not understand to the fullest detail what are the interactions between individual components that determine the shape of the coat, and therefore how the ER membrane will be deformed. This is important because the shape of the transport vesicle must be adapted to the type of cargo: COPII relies in fact on other proteins (regulators) to help form different shapes which are required for different cargoes. When individual coat components have some defects the transport system fails to do its job efficiently. In the worst cases this leads to dire consequences, with cells unable to survive. Sometimes, COPII defects only affect its ability to transport certain categories of cargo proteins: cells survive but they don't function in their every aspect, leading to genetic disease. For example, some mutations in COPII hamper ER exit of collagen precursors, leading to defects in development of skeletal and connective tissues. In order to understand the mechanisms that link assembly of coat components to determination of coat and membrane shape, and ability to transport all cargoes, we need to obtain a much clearer (higher resolution) picture of the COPII coat "in action". Cryo-electron tomography is a technique which allows 3D visualisation of biological specimens in near-native conditions. The resolution of this technique, especially when associated to in depth image processing, has leaped forward in the last few years, and today allows for views of biological events such as COPII budding which are a lot less "fuzzy" than before. In my lab, we have recently obtained one of the highest resolution reconstructions of part the coat. In this project, we aim to complete the picture by tackling the molecular interactions between coat components, and we also aim to study assemblies formed in increasingly physiological contexts. This will allow us to understand with the clearest molecular view how COPII assembled to form membrane-deforming scaffolds of various types, and how regulators can interact with COPII to modulate this process.

Impact Summary

Research in my lab will impact UK society and economy by benefiting a number of sectors: 1) The UK science and technology sector. The BBSRC funds will contribute to hiring a postdoctoral research assistant. This will help retain talented researchers in the UK, and/or attract young scientists from abroad. At the end of the three-year grant period, the postdoctoral researcher will master a number of transferable skills which will contribute to UK economic and scientific growth. 2) The drug discovery and diagnostic industries. It has become clear that cryo-EM is a powerful technique for pharmaceutical research and development, and an increasing number of UK-based companies are investing in this technology. This is testified by the recent explosion in the number of cryo-EM jobs in industry, by the increasing number of industry researchers that collaborate with academics to use this technique and participate to cryo-EM training courses, and by the recent acquisition of a number of high-end cryo-EM instruments by companies such as Glaxo-Smith-Kline or AstraZeneca. Our academic progress will directly inform technological development applicable to a number of fields in the pharmaceutical industry, which will directly impact the development of this sector. 3) Young future scientists from all backgrounds. The UK population is very diverse, and this has a tremendous potential to benefit scientific progress. As the people responsible for the upbringing of the next generation of scientists, we have a responsibility to attract the brightest people from the whole population, with a particular attention to currently under-represented groups, in particular women and people from minority ethnic backgrounds. I will continue acting as a mentor in programs aimed at young people from under-represented ethnicities, such as the destination STEMM program from the Windsor Fellowship, and to visit school and invite students to visit the lab. This will allow me to become a strong role model, especially for young girls, and to inspire them to take up a career in science and technology.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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