Establishing a blueprint for nuclear pore complex assembly

描述(申请人提供)：核孔复合体(NPC)提供了控制所有真核生物中跨核膜(NE)分子双向交换的唯一通道。从人类健康的角度来看，NPC的几个组成部分(核孔素/核蛋白)的功能受损与多种疾病有关，包括癌症、心脏病、AAA综合征以及阿尔茨海默氏症和帕金森氏症等神经退行性疾病。此外，为了传播和促进感染，病毒通常调节NUP功能。这些病理机制的广谱表明，鼻咽癌影响了广泛的基本细胞过程，尽管机制尚不清楚。此外，在发育和疾病背景下，几个nup基因被上调或下调，鼻咽癌数量发生变化。因此，更好地了解促进NPC功能的机制将可能揭示未来翻译药物的靶点。虽然作为一个领域，我们对管理核运输的基本机制有很好的了解，但仍然存在的一个主要挑战是确定从头开始的全国人大大会的机制。具体地说，人们还不知道~30个单独的核子是如何在空间和时间上协调组装成~50 MD NPC的。此外，单个核素的组装与膜曲率的产生相一致，膜曲率导致内核膜和外核膜紧密对接并最终融合，从而产生核孔。目前尚不清楚NUP组装和膜融合是如何协调的，融合机制也没有明确确定。在这项建议中，我们旨在解决理解全国人大大会的两个关键挑战。首先，我们提出了一种实验策略，旨在阐明核素在中间相期间在NE处组装的顺序。我们将通过利用酵母的遗传工具箱来产生一个系统来实现这一点，在这个系统中，我们一次快速且特定地灭活一个新合成的核蛋白，而不影响成熟的NPC。在每个目标NUP失活后，我们将综合检查其他NUP的分布，以按照组装的顺序分配上行或下行关系。整合这个数据集，我们将系统地定义组装过程中的步骤。其次，我们将使用顺序分析和候选方法来识别在NPC组装过程中产生膜曲率以支持NE孔形成的蛋白质。利用体内和体外的一系列方法，我们将测试这些候选曲率生成器是否能够直接驱动膜曲率，并在它们的曲率生成域中定位。通过这种方式，我们将为孔形成的分子机制提供重要的新线索，并直接测试膜曲率如何影响NPC组装。这项提案中概述的实验将为所有真核生物普遍存在的鼻咽癌生物发生的基本过程提供亟需的机械性洞察。

DESCRIPTION (provided by applicant): Nuclear pore complexes (NPCs) provide the sole gateways that control the bidirectional exchange of molecules across the nuclear envelope (NE) in all eukaryotes. From the perspective of human health, compromised function of several of the components of the NPC (nucleoporins/nups) is associated with diverse diseases including cancers, heart disease, triple A syndrome, and neurodegenerative diseases like Alzheimer's and Parkinson's. Further, to propagate and promote infection, viruses often modulate nup function. The wide spectrum of these pathologies suggests that the NPC impacts a broad array of essential cellular processes, although the mechanisms are poorly defined. In addition, several nup genes are up- or down- regulated, and NPC number is altered, in developmental and disease contexts. Thus, a better understanding of mechanisms that contribute to NPC function will likely reveal translational drug targets in the future. While, as a field, we have a good understanding of the underlying mechanisms governing nuclear transport, a major remaining challenge is determining the mechanism of de novo NPC assembly. Specifically, it is not understood how the ~30 individual nups are coordinately assembled in space and time to form the ~50 MD NPC. Further, the assembly of individual nups coincides with the generation of membrane curvature that leads to the close apposition and eventual fusion of the inner and outer nuclear membranes to generate a nuclear pore. It is not known how nup assembly and membrane fusion are coordinated, nor has the fusion machinery been clearly identified. In this proposal, we aim to tackle two key challenges in understanding the assembly of the NPC. First, we propose an experimental strategy designed to elucidate the order by which nups are assembled at the NE during interphase. We will achieve this by exploiting the genetic toolkit of the yeast, S. cerevisiae, to generate a system where we rapidly and specifically inactivate newly synthesized nups one at a time, leaving mature NPCs unaffected. After inactivation of each target nup, we will comprehensively examine the distribution of other nups to assign an up- or down-stream relationship in the order of assembly. Integrating this data set, we will systematically define the steps in the assembly process. Second, we will use both the order analysis and a candidate approach to identify proteins that generate membrane curvature to support pore formation in the NE during NPC assembly. Using a series of in vivo and in vitro approaches, we will test whether these candidate curvature generators are capable of directly driving membrane curvature and home in on their curvature-generating domains. In this way, we will shed significant new light onto the molecular mechanisms of pore formation and directly test how membrane curvature impacts NPC assembly. The experiments outlined in this proposal will provide much needed mechanistic insight into the essential process of NPC biogenesis, universal to all eukaryotes.