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

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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.