Understanding mechanisms of coat assembly and membrane deformation.

单元格可以比作城市来描述它们的结构和组织。细胞的公民是蛋白质，它执行维持细胞动态平衡和确保生存和增殖所需的所有活动。真核细胞组织在被称为隔室的膜所分隔的区域。这些可以被认为是精确定义的社区，在那里发生特定的活动。例如，一个被称为内质网的细胞器(缩写为ER)是制造某些类别蛋白质的地方，也是对它们的健康状况进行初步检查的地方。另一个重要的细胞器是高尔基体：这是蛋白质成熟为其完全功能的形式并被分类到它们执行工作的地方。就像在城市一样，蛋白质需要从一个隔室运输到另一个隔室--这是维持细胞功能所必需的一个基本方面。由于细胞隔间是由膜分隔的，隔间之间的交流和物质交换通过小泡进行：膜芽的小囊从装载货物的原始隔室中掐下，然后将其释放到目标隔室。连接内质网和高尔基体的运输链路被称为COPII，它将年轻的蛋白质从它们出生的地方转移到它们将完全成熟的地方。COPII是一组蛋白质，通过组装到内质网膜周围的弯曲支架中帮助内质网形成囊泡，使其萌发一个囊泡(COPII从而形成所谓的“外套”)。在使内质网细胞膜变形的同时，COPII还与内质网内需要运输的蛋白质(货物)连接，使这些蛋白质结合到形成的小泡中。内质网膜的变形遵循被膜的形状。我们对COPII机制有很好的功能理解，但我们对膜上被膜作用的看法有些模糊。特别是，我们不能非常详细地了解决定涂层形状的各个组件之间的相互作用，从而确定ER膜将如何变形。这一点很重要，因为运输囊泡的形状必须适应货物的类型：COPII实际上依赖于其他蛋白质(调节器)来帮助形成不同货物所需的不同形状。当单个涂层部件有一些缺陷时，运输系统不能有效地完成其工作。在最糟糕的情况下，这会导致可怕的后果，细胞无法存活。有时，COPII缺陷只会影响其运输特定类别货物蛋白质的能力：细胞存活，但它们并不能在各个方面发挥作用，从而导致遗传疾病。例如，COPII中的一些突变阻碍了胶原前体的内质网退出，导致骨骼和结缔组织的发育缺陷。为了了解将涂层成分的组装与涂层和膜形状的确定以及运输所有货物的能力联系起来的机制，我们需要获得更清晰(更高分辨率)的COPII涂层“作用”的图像。低温电子断层扫描是一种允许在近自然条件下对生物标本进行3D可视化的技术。这项技术的分辨率，特别是与深度图像处理相关的分辨率，在过去几年里有了飞跃，现在可以看到像COPII萌芽这样的生物事件，比以前少了很多的“模糊”。在我的实验室里，我们最近获得了部分毛皮的最高分辨率重建之一。在这个项目中，我们的目标是通过解决涂层成分之间的分子相互作用来完成这一图景，我们的目标也是研究在越来越多的生理环境中形成的组件。这将使我们能够以最清晰的分子观点了解COPII是如何组装形成各种类型的膜变形支架的，以及调节剂如何与COPII相互作用来调节这一过程。

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.