Dissecting the molecular machinery of nuclear transport

摘要：核孔复合物（NPCs）是细胞核和细胞质隔室之间通过核膜（NE）进行交换的唯一介质。核细胞质转运依赖于转运货物、同源可溶性转运因子（许多称为Kaps）和npc之间的相互作用。我们采用了一种全面的方法来定义模型真核生物酵母（酵母）中NPC的功能结构。我们鉴定了所有酵母鼻咽癌蛋白（Nups）并绘制了它们在鼻咽癌中的分布；这项工作使我们能够提出一种新的核传输“虚拟门控”机制。我们还为所有的Nup分配了折叠类型，并系统地分离了Nup子复合物，以确定它们相互作用的网络。然后，我们使用这些信息计算NPC结构的3D地图，足以解析整个NPC的分子组织。我们的工作暴露了NPC架构中的简单模块化；此外，包被囊泡和鼻咽癌结构的相似性表明它们共同的进化起源是一个祖先“原囊泡”。我们现在的目标是制作NPC的高分辨率动态地图。首先，我们将使用额外的“低果实，高回报”免疫纯化和免疫定位实验来快速改进我们的NPC地图，直到我们可以辨别出其中的Nups的形状。然后，我们将使用电子显微镜和交联技术研究重组Nups和Nup复合物，以揭示在Nups及其复合物内折叠、结构域和蛋白质是如何组织的精细细节。接下来，我们将在体外重建核胞质运输的关键反应，并在体内测试可能的机制模型，以重建Kaps及其货物穿越NPC时的运动。我们最终将协同解码这些信息，并将其转换为NPC和核传输的动态3D表示，理想情况下是原子分辨率，从而使我们能够在最基本的层面上理解NPC的起源、组装和机制。我们正在研究将物质运送到活细胞DNA上的微型机器。这些机器被称为“核孔复合物”，它允许DNA向细胞的其他部分发送指令，从而帮助调节细胞如何生存、发育，并阻止自己犯在癌细胞中看到的那种错误。我们希望了解这些机器是如何工作的，以及它们是如何在生命的早期进化中出现的。

DESCRIPTION (provided by applicant): ABSTRACT Nuclear pore complexes (NPCs) are the sole mediators of exchange across the nuclear envelope (NE) between the nuclear and cytoplasmic compartments. Nucleocytoplasmic transport depends on the interplay between transport cargoes, their cognate soluble transport factors (many termed Kaps), and NPCs. We have taken a comprehensive approach to defining the functional architecture of the NPC in the model eukaryote Saccharomyces (yeast). We identified all the yeast NPC proteins (Nups) and plotted their disposition in the NPC; this work allowed us to propose a new "virtual gating" mechanism for nuclear transport. We also assigned fold types to all the Nups and systematically isolated Nup subcomplexes to determine the network of interactions they make. We then used this information to compute a 3D map of the NPC architecture, sufficient to resolve the molecular organization of the entire NPC. Our work exposed a simple modularity in the architecture of the NPC; moreover, similarities between structures in coated vesicles and those in the NPC suggest their common evolutionary origin in a progenitor "protocoatomer". Our goal is now to produce high resolution dynamic maps of the NPC. First, we will use additional "low-fruit, high-payoff' immunopurification and immunolocalization experiments to rapidly improve our NPC map, to the point at which we can discern the shapes of the Nups in it. We will then study recombinant Nups and Nup complexes using electron microscopy and crosslinking, to reveal fine details on how the folds, domains, and proteins are organized within the Nups and their complexes. Next, we will reconstitute key reactions of nucleocytoplasmic transport in vitro, and test possible mechanistic models in vivo, in order to reconstruct the movements Kaps and their cargos make on crossing the NPC. We will finally synergistically decode this information and convert it into dynamic, 3D representations of the NPC and nuclear transport, ideally at atomic resolution, thereby allowing us to understand the origin, assembly and mechanism of the NPC at the most fundamental level. LAY SUMMARY We are studying the tiny machines that shuttle materials to the DNA in living cells. These machines, called "nuclear pore complexes", allow the DNA to send its instructions to the rest of the cell, and so help regulate how a cell lives, develops, and stops itself from making the kinds of mistakes seen in cancer cells. We wish to understand how these machines work and how they arose in the early evolution of life.