Dissecting the molecular machinery of nuclear transport

描述(申请人提供)：摘要核孔复合体(NPC)是核间和细胞质间跨核膜(NE)交换的唯一媒介。核质运输依赖于运输货物、其同源可溶性运输因子(许多称为KAP)和NPC之间的相互作用。我们已经采取了一种全面的方法在真核生物酵母(酵母)模型中定义NPC的功能结构。我们鉴定了所有的酵母NPC蛋白(NUP)，并绘制了它们在NPC中的分布；这项工作使我们能够提出一种新的核运输的“虚拟门控”机制。我们还为所有的NUP指定了折叠类型，并系统地分离了NUP亚复合体，以确定它们所形成的相互作用网络。然后，我们使用这些信息计算了NPC结构的3D地图，足以解析整个NPC的分子组织。我们的工作揭示了NPC结构中的一种简单的模块化；此外，包被囊泡中的结构与NPC中的结构之间的相似性表明，它们共同的进化起源于先祖“原癌细胞”。我们现在的目标是制作高分辨率的NPC动态地图。首先，我们将使用额外的“低果率、高回报”的免疫纯化和免疫定位实验来快速改进我们的NPC地图，以使我们能够辨别其中的核形状。然后，我们将使用电子显微镜和交联法研究重组NUP和NUP复合体，以揭示折叠、结构域和蛋白质在NUP及其复合体中是如何组织的细节。接下来，我们将在体外重建核质转运的关键反应，并在体内测试可能的机制模型，以重建KAPS及其货物通过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和核运输的动态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了解NPC的起源、组装和机制。我们正在研究将材料运送到活细胞中的DNA的微型机器。这些被称为“核孔复合体”的机器允许DNA将其指令发送到细胞的其他部分，从而帮助调节细胞的生存、发育，并阻止自己犯下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.