High Resolution Cryo-Electron Microscopy of Clathrin Cage Networks

在低温电子显微镜中，新型直接电子探测器的卓越信号灵敏度使结构测定领域发生了革命性的变化，使得无需使用X射线结晶学就可以获得具有挑战性的靶的亚结构，例如膜蛋白和核糖体。笼状蛋白介导的内吞作用是一种令人着迷的机械现象，它驱动分子选择性地内化到细胞中。为了更好地发挥作用，网状蛋白介导的内吞作用需要准确和及时地组装网状结构的网状结构，并与20多个接头蛋白组成的网络进行协调，形成包被的囊泡，该包被的小泡能够从细胞外选择分子输送到特定的目的地。本项目的目的是通过利用低温电子显微镜对网状蛋白笼状复合体进行高分辨率结构分析，阐明参与网状蛋白网络的蛋白质与其接头蛋白如何相互作用以实现网状蛋白包膜囊泡的形成。高分辨率3D冷冻电子显微镜将被用来显示与笼状蛋白骨架结合的接头蛋白，以及生物物理方法，如动态光散射、时间分辨荧光各向异性和恒温滴定量热法，以研究骨架蛋白-接头相互作用如何形成功能性涂层囊泡网络。

In cryo-electron microscopy, the superior signal sensitivity of new direct electron detectors has revolutionised the field of structure determination allowing sub-4Å structures of challenging targets such as membrane proteins and ribosomes to be obtained without using X-ray crystallography. Clathrin-mediated endocytosis is a fascinating mechanical phenomenon that drives the selective internalisation of molecules into cells. In order to work properly, clathrin-mediated endocytosis requires accurate and timely assembly of a clathrin lattice and coordination with a network of more than 20 adaptor proteins to form a coated vesicle which will be able to select molecules from the outside of the cell for delivery to specific destinations.The aim of this project is to elucidate how the proteins involved in the network of clathrin and its adaptor proteins interact to achieve clathrin-coated vesicle formation by exploiting cryo-electron microscopies capability to carry out high resolution structural analysis of clathrin cage complexes. High resolution 3D cryo-electron microscopy will be used to visualise adaptor proteins binding to clathrin cages as well as biophysical approaches such as dynamic light scattering, time-resolved fluorescence anisotropy and isothermal titrating calorimetry in order to investigate how clathrin-adaptor interactions result in formation of a functional coated vesicle network.