Moonlighting functions of endocytic proteins during the cell cycle

Cells can be thought of as islands - they are closed to the outside world because they have a plasma membrane that doesn't let anything through. However, cells have evolved tiny portals for things to enter in a tightly regulated way. These portals are working all the time, but they are shut down when the cell needs to divide. It is important that no mistakes are made at this time. Otherwise the cell could die or it could start to grow in an unusual way leading to disease e.g. cancer. Nobody knows how this shutdown happens. We want to study the portals using a machine that can tell us all the pieces that make up a portal. If we compare portals from normal cells with those that are dividing this will tell us which pieces are missing in dividing cells. We can then find out how the cell takes these pieces away at the start of cell division. We think that this work is important to do. These portals are hijacked by viruses: they use portals as a way to infect cells. If we knew how the cell shuts down the portals, we could think of ways to switch them off using drugs. These drugs could be used as anti-viral treatments.

Clathrin-coated vesicles (CCVs) are formed continuously in non-dividing cells. When the cell enters mitosis, membrane trafficking is shut down. This phenomenon has been acknowledged for almost fifty years, but the mechanism behind the shutdown has been enigmatic. It is thought that certain components of the clathrin coat are phosphorylated during mitosis and this prevents their engagement with the network of proteins responsible for driving CCV formation. In addition, there is an emerging concept in cell biology of proteins with alternative functions during mitosis. In this project, we will investigate the link between those proteins that are missing from coats in mitotic cells, those that are phosphorylated and those that carry out alternative functions. We propose a systematic and quantitative proteomic study of clathrin-coated membranes from cells in interphase and in mitosis. This will allow us to identify the proteins that are less abundant in clathrin coats from mitotic cells. Using live-cell imaging, we will determine at what stage in the cell cycle these proteins cease to be recruited to the coat and also investigate where in the cell the protein is localised during this time. Finally, we will discover whether these proteins are phosphorylated and whether or not this modification is responsible for the mitotic shutdown of CCV formation.

The major stakeholders impacted by this research are: 1. International scientific community: cell biologists and biochemists 2. Local scientific community: systems biology research in Liverpool 3. Commercial sector Our strategy for engaging with these stakeholders is outlined in the Impact Plan.