Quantitative analysis of the assembly and disassembly of clathrin cages.

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.

Clathrin rapidly and reversibly forms large cage structures of varying sizes. These properties are exploited by eukaryotic cells to encapsulate endocytic transport vesicles and coordinate their formation, contents selection and delivery to target locations. We have a sensitive assay for clathrin disassembly which has allowed us to monitor clathrin disassembly with excellent time resolution. Kinetic analysis of these results reveals a model for the action of Hsc70 in which three Hsc70 molecules act in sequence to release a clathrin triskelion. We aim to quantitate and define the assembly and disassembly of clathrin cages through the following specific objectives:

1. To obtain molecular level information on the precise way in which domains of Hsc70 and auxilin carry out this mechanism. In order to do this we have created a series of mutants of Hsc70 and auxilin which contain only a single cysteine residue and to which we will attach fluorescent labels. This will provide us with a set of site-specific fluorescent probes with which we can monitor, at the domain level, the binding interactions and protein conformational changes which occur during clathrin disassembly.

2. To investigate the mechanism by which clathrin triskelions assemble both through pH jump and addition of the adaptor protein, AP2. By analysing stopped flow light scattering data of the assembly process we will be able to evaluate potential mechanisms, obtain kinetic rate constants for triskelion interactions during cage assembly and then probe how AP2 influences these rate constants.

3. To investigate how the mechanical properties of the clathrin triskelion determine successful assembly into clathrin cages. We have been successful in imaging clathrin triskelions 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 and calculate the rigidity of clathrin triskelions as they associate.

Clathrin-mediated endocytosis plays a central role in multiple cellular functions including nutrient uptake, synaptic vesicle recycling, signaling, maintenance of cell polarity and development. In addition, the endocytic apparatus is used by some viruses (notably HIV) and bacteria to gain entry into cells and there is accumulating evidence that mutations or differences in expression levels in endocytic proteins are associated with a wide range of diseases including neurodegenerative disease and cancer. Given the importance of clathrin-mediated endocytosis to health and disease, the understanding that we will gain in this project on the principles of clathrin coat assembly and disassembly will enable us to learn how to tackle disease-causing malfunctions of this system. In addition, the knowledge that we will generate concerning the ability of a cell to carry out this precise remodelling of membranes will help synthetic biologists who wish to devise systems for controlling such events outside the cell.

The immediate beneficiaries of this work will include ACADEMIC SCIENTISTS who will use the new knowledge that is gained and the new techniques that are developed and alter their own research and development activities in light of this.

The insights we uncover will be published in journals of the highest possible calibre, thus sustaining the reputation of the UK as a world leader in scientific enquiry. Learned bodies, such as the Biochemical Society and the British Society for Cell Biology will benefit because they will be able to communicate this work to the wider scientific community and the public through their public engagement activities.

The EARLY CAREER RESEARCHER associated with the project will benefit from collaborations across the disciplines of biochemistry, microscopy, biophysics and modelling and from the associated skills to accomplish this that they will gain. These skills will transfer into their future careers in whatever sector they work. 'UK plc.' will benefit from such well-trained cross-disciplinary scientists who will be suited to many avenues of research.

INDUSTRIAL SCIENTISTS who are interested in how proteins control the shape of membranes and those interested in controlling the uptake and release of specific molecules e.g. as part of a drug delivery system, will also benefit from detailed understanding of how this very process is achieved in biology. This impact will take place as soon as the knowledge is disseminated e.g. through attendance of the next BioNano Collaborative International meeting in 2013.

The academics and early stage researcher will continue their programme of SCIENCE COMMUNICATION WITH THE GENERAL PUBLIC, engaging with local schools, local and national media, science fairs, IGGY (International Gateway for Gifted Youth), MoleClues, open days etc. thus benefiting the level of education and of science debate outside academia as well as within. Encouraging scientists to take part in such activities early in their careers will establish habits and expertise for benefitting the general public that will stay with them for their careers. This impact will be immediate as well as over the next few decades.