Understanding mechanisms of coat assembly and membrane deformation.

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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.