Integrated microscopy approach to protein assembly on and in membranes

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Proteins are key building blocks and the working horses of the living cell. They generate energy, make muscles contract, and translate light in our eyes into an image as perceived by our brain, to name but a few of many examples. In many cases, proteins need to form larger assemblies to carry out their biological function. Incorrect assembly causes biological malfunction and disease. Hence there is a general interest to understand how proteins form such larger assemblies. However, though we have an extensive toolkit to determine the structures of proteins and their assemblies, it is much harder to trace the processes by which these assemblies are being built.In this project, we will therefore develop new methodology to visualise protein assembly at length and time scales that will enable us to determine how such processes take place. Because the therefore required resolution can not be achieved by one technique alone, we will use a combination of different microscopy techniques: Single-molecule fluorescence microscopy, which can track individual labelled proteins as they move about; atomic force microscopy, which can visualise protein assemblies as they are being formed; and electron microscopy, which can provide static snapshots of the structure of proteins and protein assemblies.To test this correlative and integrated microscopy approach, we will apply it to a protein (named perforin) that is used by the immune system to attack virally infected and cancerous cells in the human body, essentially by perforating the membranes that protect cells from their environment. Perforin drills holes in these membranes by forming assemblies of several tens of proteins that span the membrane.We aim to understand the mechanisms of membrane pore formation by these proteins, out of a fundamental interest in how this nanometre-scale machinery works, to better understand diseases or enhanced vulnerability to cancer caused by malfunctioning perforin, and because mechanistic understanding can facilitate the design of drugs that prevent such pores from being formed when the immune response needs to be suppressed transplant, for example during organ transplantation.