High Resolution Assembly Structure of the Nuclear Pore Complex

 描述（申请人提供）：细胞核是真核细胞的内部避难所，将遗传信息保存在核被膜（NE）中。NE由两个同心的膜双层在特定位点融合形成圆形开口，每个开口被核孔复合物（NPC）占据。作为进出细胞核的唯一通道，NPC在细胞内稳态中起着关键作用，但它们不仅仅是运输复合物。我们开始意识到NPC也可以作为组织细胞核的参考点，并在基因调控中发挥关键作用。该研究计划的重点是确定巨大的~40-80 MDa NPC的分子结构。实现这一目标将提供一个结构基础，询问其在运输，基因调控和核组织的无数功能。重要的是，NPC及其成分的异常功能是许多人类疾病的主要原因，包括白血病、其他癌症、自身免疫性疾病、心肌病和各种病毒感染。NPC是一种模块化结构，由约30种不同的蛋白质组成，称为核孔蛋白，它们围绕中心八重旋转轴排列成多个拷贝。2-10个核孔蛋白的亚复合物分层构建NPC。这些亚复合物中的两个，异聚Y-和Nic 96-复合物，组织了大多数结构核孔蛋白，并建立了NPC的稳定支架。最近，不同实验室对这些亚复合物的晶体学分析结合冷冻电子断层扫描重建，导致了第一个模糊的组装模型。在这里，我们建议充分结构表征支架组件的NPC。通过生物化学和细胞生物学方法的补充，我们假设我们将生成数据来构建NPC的详细结构，NPC是真核细胞中最大的组装结构。该提案的核心是使用X射线晶体学和电子显微镜技术的组合，以近原子分辨率建立NPC的主要构建模块。本研究的第二个重点是使用实验方法建立所有核孔蛋白的相互作用组。这些信息对于建立一个强大的NPC模型至关重要。NPC的这种3D再现然后可以用于在详细的分子水平上询问NPC功能。

 DESCRIPTION (provided by applicant): The nucleus is the inner sanctuary of the eukaryotic cell, keeping the genetic information enclosed by the nuclear envelope (NE). The NE consists of two concentric membrane bilayers fused at specific sites to form circular openings, each occupied by a nuclear pore complex (NPC). As the only gateway into and out of the nucleus, NPCs play a critical role in cellular homeostasis, but they are more than just transport complexes. We are beginning to appreciate that the NPC also serves as a reference point to organize the nucleus and plays a pivotal role in gene regulation. This research program focuses on determining the molecular architecture of the enormous ~40-80 MDa NPC. Attaining this goal will provide a structural basis to interrogate its myriad functions in transport, gene regulation, and nuclear organization. Importantly, aberrant function of the NPC and its constituents is a primary cause of many human diseases, including leukemia, other cancers, autoimmune diseases, cardiomyopathies, and a variety of viral infections. The NPC is a modular structure, composed of ~30 different proteins, known as nucleoporins that arrange in multiple copies around a central eightfold rotational axis. Subcomplexes of 2-10 nucleoporins hierarchically build up the NPC. Two of these subcomplexes, the heteromeric Y- and Nic96-complexes, organize the majority of architectural nucleoporins and establish the stable scaffold of the NPC. Crystallographic analysis of these subcomplexes by different labs combined with cryo-electron tomographic reconstitution has recently led to a first, vague assembly model. Here, we propose to fully structurally characterize the scaffold components of the NPC. Complemented by biochemical and cell biological methods, we hypothesize that we will generate the data to build a detailed structure of the NPC, the largest assembly structure in the eukaryotic cell. The proposal is centered on establishing the major building blocks of the NPC at near-atomic resolution, using a combination of X-ray crystallographic and electronmicroscopic techniques. A second focus of this study is to establish the interactome of all nucleoporins using experimental methods. This information will be critical in order to build a robust model of the NPC. Such a 3D rendition of the NPC can then be used to interrogate the NPC functions at a detailed, molecular level.