Establishing a blueprint for nuclear pore complex assembly

描述（由申请人提供）：核孔复合物（NPC）提供了控制所有真核生物中跨核膜（NE）的分子双向交换的唯一通道。从人类健康的角度来看，NPC的几种组分（核孔蛋白/nups）的功能受损与多种疾病相关，包括癌症、心脏病、三A综合征和神经退行性疾病如阿尔茨海默病和帕金森病。此外，为了传播和促进感染，病毒经常调节nup功能。这些病理的广泛范围表明NPC影响广泛的基本细胞过程，尽管机制定义不清。此外，在发育和疾病背景下，几个nup基因上调或下调，并且NPC数量改变。因此，更好地了解有助于NPC功能的机制可能会在未来揭示翻译药物靶点。虽然，作为一个领域，我们有一个很好的理解的基本机制，管理核运输，一个主要的挑战是确定从头NPC组装的机制。具体而言，尚不清楚约30个单独的nup如何在空间和时间上协调组装以形成约50个MD NPC。此外，单个核团的组装与膜曲率的产生一致，膜曲率导致内核膜和外核膜的紧密并置和最终融合以产生核孔。目前尚不清楚nup组装和膜融合是如何协调的，也没有明确的融合机制。在本提案中，我们旨在解决理解人大会议的两个关键挑战。首先，我们提出了一个实验策略，旨在阐明nups组装在NE在相间的顺序。我们将通过利用酵母S.酿酒酵母，以产生一个系统，在这个系统中，我们快速和特异性地一次一个地抑制新合成的nups，使成熟的NPC不受影响。在灭活每个目标nup后，我们将全面检查其他nup的分布，以按组装顺序分配上游或下游关系。整合这些数据集，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产生膜曲率以支持NPC组装期间NE中孔形成的蛋白质。使用一系列的体内和体外方法，我们将测试这些候选曲率发生器是否能够直接驱动膜曲率和家庭在其曲率生成域。通过这种方式，我们将为孔形成的分子机制提供重要的新见解，并直接测试膜曲率如何影响NPC组装。在这个建议中概述的实验将提供急需的机制洞察NPC生物发生的基本过程，普遍的所有真核生物。

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.