Integrated microscopy approach to protein assembly on and in membranes

蛋白质是活细胞的关键构件和工作马匹。它们产生能量，使肌肉收缩，并将我们眼中的光转化为我们大脑所感知的图像，仅举几例。在许多情况下，蛋白质需要形成更大的组件来执行其生物功能。不正确的组装会导致生物功能障碍和疾病。因此，人们普遍有兴趣了解蛋白质是如何形成如此大的组装体的。然而，尽管我们有一个广泛的工具包来确定蛋白质及其组装的结构，但要跟踪这些组装的过程要困难得多。因此，在这个项目中，我们将开发新的方法来可视化蛋白质组装的长度和时间尺度，使我们能够确定这些过程是如何发生的。由于所需的分辨率不能单独通过一种技术实现，我们将使用不同的显微镜技术的组合：单分子荧光显微镜，可以跟踪单个标记的蛋白质，因为它们移动;原子力显微镜，可以可视化蛋白质组装，因为它们正在形成;和电子显微镜，它可以提供蛋白质和蛋白质组装体结构的静态快照。为了测试这种相关和综合的显微镜方法，我们将把它应用于一种蛋白质（名为穿孔素），这种蛋白质被免疫系统用来攻击人体内受病毒感染的细胞和癌细胞，基本上是通过穿孔保护细胞免受环境影响的膜。穿孔素通过形成跨越膜的数十种蛋白质的组装体在这些膜上钻孔。我们的目标是了解这些蛋白质形成膜孔的机制，出于对这种纳米级机器如何工作的根本兴趣，以更好地了解穿孔素故障引起的疾病或对癌症的易感性增强，并且因为对机理的理解可以促进药物的设计，所述药物在需要抑制免疫反应时，例如在器官移植期间，防止形成这种孔。

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.