Exploiting direct electron detection to resolve protein-protein interactions in clathrin-mediated endocytosis

网格蛋白介导的胞吞作用在多种细胞功能中起核心作用，包括营养摄取、突触囊泡循环、信号传导、维持细胞极性和发育。此外，一些病毒（特别是艾滋病毒和流感病毒）和细菌利用内吞装置进入细胞，越来越多的证据表明，内吞蛋白表达水平的突变或差异与包括神经退行性疾病和癌症在内的多种疾病有关。网格蛋白介导的胞吞作用是通过细胞膜形成囊泡来捕获货物分子。这个过程是由一个蛋白质网络控制的，其中包括网格蛋白和一组与网格蛋白结合的衔接蛋白。网格蛋白组装形成一个多面体笼，与它的接头蛋白一起，在囊泡周围形成一层外壳。然后，囊泡从膜上分离，移动到细胞内的某个位置，运送其内容物。这么多不同的接头蛋白是如何与单一蛋白协调，从而使这个细胞邮政系统实现其功能的，这仍然是一个谜。本研究旨在通过两种主要方法阐明一种或多种衔接蛋白如何与网格蛋白笼结构相互作用。首先，也是最重要的是，电子显微镜令人兴奋的新发展提供了一个独特的机会来推进分辨率，我们可以对这些笼子进行成像。高分辨率图像将揭示组装的多蛋白复合物的详细结构，并显示组分之间相互作用的位置。其次，通过使用先进的生物物理技术，我们将测量一组接头蛋白如何单独或成对地与网格蛋白结合，以找出不同的接头蛋白是否使用相同或不同的结合位点，或位点组合，以及接头蛋白之间是否存在竞争。第三，生物物理数据将通过与多个接头蛋白结合的网格蛋白笼成像并将其与与单个接头结合的结构进行比较来扩展。通过分析不同的图像，我们将获得关于这些笼如何组装和拆卸以及多个接头蛋白在这些过程中的作用的非常详细的3D信息。因此，我们将获得网格蛋白外壳如何指定和参与细胞中如此多的关键功能的关键信息。

Clathrin-mediated endocytosis plays a central role in multiple cellular functions including nutrient uptake, synaptic vesicle recycling, signaling, maintenance of cell polarity and development. In addition, the endocytic apparatus is used by some viruses (notably HIV and influenza) and bacteria to gain entry into cells and there is accumulating evidence that mutations or differences in expression levels in endocytic proteins are associated with a wide range of diseases including neurodegenerative disease and cancer. Clathrin-mediated endocytosis operates through formation of a vesicle from the cell's membrane trapping cargo molecules inside. This process is controlled by a network of proteins which include clathrin and a set of adaptor proteins which bind to clathrin. Clathrin assembles to form a polyhedral cage which, together with its adaptor proteins, forms a coat around the vesicle. The vesicles then detach from the membrane and move to a location inside the cell to deliver its contents. It remains a mystery how so many different adaptor proteins coordinate with a single protein so that this cellular postal system can achieve its function. This proposal aims to elucidate how one or more adaptor proteins interact with clathrin cage structures by using two main approaches. First, and most importantly, exciting new developments in electron microscopy have provided a unique opportunity to push forward the resolution with which we can image these cages. High resolution images will reveal the detailed structures of the assembled multi-protein complex and show where components interact with one another. Secondly, by using advanced biophysical techniques we will measure how a set of adaptor proteins bind to clathrin individually and in pairs, to find out whether different adaptors use the same or different binding sites, or a combination of sites and if there is competition between adaptor proteins. Thirdly, the biophysical data will be extended by imaging clathrin cages bound to multiple adaptor proteins and comparing these to structures bound to single adaptors. By analysing difference images we will obtain highly detailed 3D information on how these cages assemble and disassemble and on the roles of multiple adaptor proteins in these processes. As a result, we will obtain key information on how clathrin coats specify and are involved in so many critical functions in cells.