Quantitative analysis of the assembly and disassembly of clathrin cages.

网格蛋白是一种快速可逆地形成大小不一的大笼状结构的蛋白质。细胞利用这些特性，通过网格蛋白介导的内吞作用来吸收和运输它们生存所需的物质。网格蛋白介导的胞吞作用在多种细胞功能中起着核心作用，包括营养摄取、神经细胞功能、细胞内通讯和生物体发育。此外，一些病毒（特别是艾滋病毒）和细菌利用这种装置进入细胞，越来越多的证据表明，参与内吞作用的蛋白质与包括神经退行性疾病和癌症在内的多种疾病有关。考虑到网格蛋白介导的内吞作用对健康和疾病的重要性，如果我们要学习如何解决该系统的致病故障，了解网格蛋白外壳组装和拆卸的原理是至关重要的。所使用的载体是由细胞膜通过许多不同蛋白质网络的作用形成的，包括网格蛋白，它在运输囊泡周围形成一个专门的外壳，形成“网格蛋白包裹的囊泡”。网格蛋白包被囊泡的组装和拆卸对它们的生命周期至关重要，但关于这一过程如何工作的许多细节尚不清楚。分解是由两种相对较小的蛋白质处理的，Hsc70是一种分子伴侣或“辅助”蛋白质，而auxilin/GAK是Hsc70的辅助因子。它们的作用是分解由于多个网格蛋白分子聚集在囊泡周围而产生的组装。网格蛋白分子比Hsc70和辅助蛋白大得多，并且具有有趣的三条腿外观，这使得网格蛋白组合具有不规则足球的外观和几何形状，具有五边形和六边形的面。我们的目标是找出这些小分子是如何快速而安全地拆除网格蛋白外壳的，并了解单个三足网格蛋白分子的结合如何导致笼状结构的形成。我们将采用三种策略来研究网格蛋白的组装和拆卸：首先，我们制作了一个可以在已知位点标记的Hsc70和辅助蛋白分子的工具包。通过使用这些标签观察网格蛋白的分解，我们将能够看到辅助蛋白和Hsc70的哪些部分对于将网格蛋白分子分开更重要。其次，我们将通过分析笼状结构在形成时散射光的方式来研究网格蛋白三螺旋组装的相关机制。这将使我们能够测量哪些因素，特别是其他涂层成分，影响组装，数据分析将帮助我们拼凑出笼形成的事件顺序。我们的最终目的是获得网格蛋白三旋翼的机械性能的详细测量。我们已经能够使用高度新颖和最先进的高速原子力显微镜技术实时拍摄网格蛋白三棱的运动。因此，我们将能够直接观察笼形结构的组装，计算笼形结构的刚度，并了解笼形结构的机械性能如何决定笼形结构的成功组装。

Clathrin is a protein which rapidly and reversibly forms large cage structures of varying sizes. These properties are exploited by cells in order to absorb and transport the substances they need to survive through clathrin-mediated endocytosis. Clathrin-mediated endocytosis plays a central role in multiple cellular functions including nutrient uptake, nerve cell function, communication within the cell and organism development. In addition, such apparatus is used by some viruses (notably HIV) and bacteria to gain entry into cells and there is accumulating evidence that proteins involved in endocytosis are associated with a wide range of diseases including neurodegenerative disease and cancer. Given the importance of clathrin-mediated endocytosis to health and disease, understanding the principles of clathrin coat assembly and disassembly is vital if we are to learn how to tackle disease-causing malfunctions of this system. The vehicles which are used are formed from cell membranes through the action of a network of many different proteins, including clathrin, which form a specialised coat around transport vesicles to form 'clathrin-coated vesicles'. Assembly and disassembly of clathrin-coated vesicles is essential for their life cycle and yet many details about how this process works are not understood. Disassembly is handled by two relatively small proteins, Hsc70 which is a molecular chaperone or 'helper' protein and auxilin/GAK, which is a cofactor for Hsc70. Their role is to take apart the assembly which has been created as a result of multiple clathrin molecules coming together around the vesicle. The clathrin molecules are much larger than Hsc70 and auxilin and have an intriguing three-legged appearance which gives clathrin assemblies the appearance and geometry of an irregular football, with pentagonal and hexagonal faces. Our aim is to find out how these smaller molecules dismantle the clathrin coat speedily and without mishaps and to understand how association of individual three-legged clathrin molecules leads to formation of cage structures.We will adopt three strategies for investigating clathrin assembly and disassembly: First, we have made a tool-kit of Hsc70 and auxilin molecules which can be labelled at known sites. By watching clathrin disassembly using these labels we will be able to see which parts of auxilin and Hsc70 are more important for pulling the clathrin molecules apart. Second, we will investigate the related mechanism by which clathrin triskelions assemble by analysing the way in which the cages scatter light when they form. This will allow us to measure what factors, particularly other coat components, influence assembly and data analysis will help us piece together an order of events for cage formation. Our final aim is to obtain detailed measurements of the mechanical properties of the clathrin triskelion. We have been able to image individual clathrin triskelions as they move in real time using the highly novel and state-of-the-art technique of high-speed atomic force microscopy. As a result we will be able to observe cage assembly directly, calculate the rigidity of clathrin triskelions as they associate and understand how the mechanical properties of the clathrin triskelion determine successful assembly into clathrin cages.