Quantitative analysis of the assembly and disassembly of clathrin cages.

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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.