Hacking the molecular logic of clathrin coat formation

Every cell is enclosed by its plasma membrane. This is a protective barrier that controls what can enter or leave. Large molecules (cargo) can get into the cell using clathrin-mediated endocytosis. This works typically by a receptor on the cell surface grabbing hold of cargo and then a "pit" made of a protein called clathrin forming on the inside. The pit deepens and eventually buds off to make a tiny delivery bubble inside the cell called a clathrin-coated vesicle. This process has been studied a lot by scientists, because it is very important for lots of things that cells do: how they eat, divide and move. It is also hijacked by some viruses so that they gain access and infect the cell! If we could control this process, we would be able to do things like sending medicines into the cell or changing how cells move or divide by manipulating endocytosis. What is stopping us from doing this is a missing piece of the puzzle: how clathrin assembles the pit. We have worked out how one protein, which provides a link from the receptor to clathrin, influences the assembly; and we have also developed ways to control this event. However humans have more than ten of these proteins, and they probably work in different ways. In this project we want to understand the rules of how these natural proteins work during endocytosis. We will then use this information to design a synthetic protein that works with very high efficiency. This will allow us to assemble clathrin-coated vesicles as quickly as possible. This "super endocytosis" system will be used to manipulate cell biology by changing endocytosis and also to send foreign molecules into cells.

Clathrin-mediated endocytosis (CME) is the major pathway of entry into eukaryotic cells. CME controls many cellular processes from metabolism and signalling to organelle maintenance and cell motility. Understanding how CME works is therefore of wide interest because it influences many areas of cell biology. This pathway has not been fully exploited for practical applications. For example, an engineered form of CME could be used to manipulate cell biological processes or to allow uptake of nanoparticles into cells. A gap in our knowledge is how adaptors link cargo to the clathrin cage *and* drive assembly of the pit. This event is key to engineering endocytosis for practical applications. In this project, we plan to decipher the molecular logic of this event using a combination of structural biology (cryoEM) and cell biology. We will study ten human proteins that are thought to drive clathrin assembly but do so in a variety of ways. By unpicking these assembly rules, we aim to design a synthetic protein that will do this function without cross-talk to other cellular systems. We have previously shown that endocytosis can be triggered using adaptor fragments in live cells. The development of synthetic protein will allow us to further develop this system and refine it for practical applications. This system will then be used to change cell adhesion through modulating the cell surface population of integrins and thereby tuning extracellular matrix interactions. Ideally, our synthetic protein will outperform endogenous clathrin assembly proteins and will be used to internalise difficult cargo such as particles that, through steric hinderance, are challenging for cells to endocytose.