Award details

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

Reference	BB/N008391/1
Principal Investigator / Supervisor	Professor Corinne Smith
Co-Investigators / Co-Supervisors	Professor Alexander Cameron
Institution	University of Warwick
Department	School of Life Sciences
Funding type	Research
Value (£)	427,889
Status	Completed
Type	Research Grant
Start date	01/06/2016
End date	31/05/2019
Duration	36 months

Abstract

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.

Summary

Impact Summary

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 chemistryand 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.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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