Exploiting direct electron detection to resolve protein-protein interactions in clathrin-mediated endocytosis

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 influenza) 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. Clathrin-mediated endocytosis operates through formation of a vesicle from the cell's membrane trapping cargo molecules inside. This process is controlled by a network of proteins which include clathrin and a set of adaptor proteins which bind to clathrin. Clathrin assembles to form a polyhedral cage which, together with its adaptor proteins, forms a coat around the vesicle. The vesicles then detach from the membrane and move to a location inside the cell to deliver its contents. It remains a mystery how so many different adaptor proteins coordinate with a single protein so that this cellular postal system can achieve its function.

This proposal aims to elucidate how one or more adaptor proteins interact with clathrin cage structures by using two main approaches. First, and most importantly, exciting new developments in electron microscopy have provided a unique opportunity to push forward the resolution with which we can image these cages. High resolution images will reveal the detailed structures of the assembled multi-protein complex and show where components interact with one another. Secondly, by using advanced biophysical techniques we will measure how a set of adaptor proteins bind to clathrin individually and in pairs, to find out whether different adaptors use the same or different binding sites, or a combination of sites and if there is competition between adaptor proteins. Thirdly, the biophysical data will be extended by imaging clathrin cages bound to multiple adaptor proteins and comparing these to structures bound to single adaptors. By analysing difference images we will obtain highly detailed 3D information on how these cages assemble and disassemble and on the roles of multiple adaptor proteins in these processes. As a result, we will obtain key information on how clathrin coats specify and are involved in so many critical functions in cells.

Endocytosis is a fundamental eukaryotic process, which underpins a wide variety of vital cellular functions including nutrient uptake, signalling, development and synaptic transmission. The interaction of clathrin with adaptor proteins is critical to clathrin-mediated endocytosis yet we still do not understand in molecular detail how single, still less multiple adaptor proteins interact with clathrin cages. Adaptor proteins are known to carry multiple short clathrin-interaction motifs as part of extensive intrinsically-disordered domains through which they bind to the polyhedral cage structure. These binding interactions with the complete cage structure have never before been defined and progress has been limited because neither cages nor intrinsically-disordered domains are amenable to crystallography. This proposal aims to elucidate how individual or pairs of adaptor proteins interact with whole clathrin cage structures by exploiting exciting new developments in the field of 3D cryo-electron microscopy to obtain the three-dimensional structure of clathrin cage complexes at the highest resolution possible. High resolution structures will be complemented by competition binding analysis and dynamic functional assays to quantify the contribution of key clathrin binding motifs to each interaction. We have three specific objectives:
1. Obtain high-resolution 3D cryo-electron microscopy structures of clathrin cages bound to single endocytic adaptor proteins
2. Obtain high-resolution 3D structures and biophysical data for clathrin cages in stable complexes with multiple adaptor proteins
3: Investigate the role of adaptor protein clathrin binding motifs using mutagenesis and functional assays
Fulfillment of these objectives will reveal the critical structures for adaptor protein function in cage assembly, establish the distribution of adaptor binding sites around the clathrin cage and shed light on the role of intrinsic disordered domains in endocytosis.

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 (e.g. HIV, influenza) 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, some psychiatric conditions and cancer. Given the importance of clathrin-mediated endocytosis to health and disease, the understanding that we will gain in this project on the interactions that clathrin makes with other coat proteins will enable us to learn how to tackle disease-causing malfunctions of this system. For example through development of specific inhibitors of interactions between clathrin and a particular adaptor protein. 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. For example, drug delivery is a highly important area of pharmaceutical development. During endocytosis, specific cargoes are absorbed by the cell, taken up into vesicles formed through the clathrin-adaptor assembly process and delivered to the correct destination. Insights gained from studying the biology of the natural delivery system exemplified by clathrin-mediated endocytosis will help those attempting to create synthetic drug delivery systems, delivering a major impact on health, economics and society.

The EARLY CAREER RESEARCHER associated with the project will benefit from collaborations across the disciplines of biochemistry, structural biology, biophysics and modelling and from the excellent training that they will gain in sought after skills such as cryo-electron microscopy, protein chemistry and data analysis. 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.

IMAGING COMPANIES associated with this project which include Jeol U.K. Ltd, Gatan and FEI will benefit from the high profile results of this project which will highlight the capabilities of the imaging equipment used in our work. The success of this project is likely to attract considerable interest due to the unusual shape and technical challenges offered by our sample, which will make the work highly desirable for use as a case study by these companies.

INDUSTRIAL SCIENTISTS such as those in the biotech and pharmaceutical industries who need to understand how proteins control the shape of membranes and how uptake and release of specific molecules can be achieved 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.

This research is likely to provide additional LONGER TERM BENEFITS, reducing cost to the NHS, increasing commercial success for companies successful in designing drugs based on this knowledge and improving the health and well being of the general public.

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), 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 both immediate and far-reaching.