Regulation of Nuclear Pore Complex Assembly

描述（由申请人提供）：核孔复合物（NPC）形成进入和离开细胞核的唯一位点。这包括小分子的扩散和大蛋白质和RNA的选择性主动运输。适当的NPC生物发生对于细胞分裂、分化和对代谢活动变化的反应至关重要。在分子水平上理解NPC组装将是设计抑制细胞生长和基因表达的策略的关键，例如在肿瘤发生中。然而，从头插入的NPC到完整的核膜（NE）的分子途径在很大程度上是不确定的。该领域最突出的问题涉及到如何描绘核孔的形成是执行和调节。协调至少30种不同NPC蛋白（Nups）和多孔膜蛋白（Pom）之间相互作用的途径是未知的。本项目旨在揭示鼻咽癌转运机制的分子生物学机制。我们的具体目标将分别利用侵略性酵母S。酿酒酵母遗传学/生物化学/细胞生物学为基础的方法直接耦合国家的最先进的显微镜和生物化学在后生动物细胞和非洲爪蟾卵提取物在体外组装系统。只有通过两个PI的全面合作，才能实现多个模型系统优势的创新融合。我们推测从头组装是一个逐步的过程。我们的第一个具体目标将解决NE和染色质的早期组装事件。使用一套新的酵母NPC装配突变体和体外非洲爪蟾试验，我们将确定的基本NUPS和装配因子，针对染色质，外和/或内核膜。将直接测试靶向机制和在组装中的作用。第二个目标将集中于外核膜和内膜融合的机制，即穿过NE形成孔，并建立在我们最近发现的NPC组装所需的ER/NE蛋白和我们在爪蟾提取物中的新孔融合测定的基础上。我们推测，高度弯曲的孔膜形成的行动，通过短暂的关联的网织蛋白稳定，并保持招聘的Nup 107 -160/Nup 84复合物形成的膜涂层。在第三个目标中，我们将分析生物发生过程中连续步骤之间的协调。将通过扫描电子显微镜检查组装中间体，并从在不同步骤中停滞的酵母突变体细胞中纯化。我们还将通过真实的实时单孔分辨率的NPC组装的荧光成像来确定膜孔形成后后生动物Nup募集的顺序。总之，这些研究将定义完整内斯中NPC生物发生的膜融合机制和自组装步骤的顺序。 公共卫生相关性：蛋白质和RNA在细胞核和细胞质之间的运输对于正常细胞功能的各个方面都是必不可少的，并且需要大的蛋白质机器（核孔复合物）来允许货物移动。贩运在某些疾病状态下受到干扰，例如癌症和病毒感染，还有一些遗传性疾病与编码运输因子和核孔复合体的基因的变化有关。核孔复合物如何组装和功能的知识将是重要的，为寻找治疗策略，以调节疾病状态和中度病毒增殖/发病机制的核运输。

DESCRIPTION (provided by applicant): Nuclear pore complexes (NPCs) form the only sites for entry and exit from the nucleus. This includes the diffusion of small molecules and the selective, active transport of large proteins and RNA. Proper NPC biogenesis is critical for cell division, differentiation, and responses to changes in metabolic activities. Understanding NPC assembly at the molecular level will be key for designing strategies to inhibit cell growth and gene expression, for example in oncogenesis. However, the molecular pathway for de novo insertion of NPCs into the intact nuclear envelope (NE) is largely undefined. The most outstanding question in the field involves delineating how nuclear pore formation is executed and regulated. The pathway for coordinating interactions between at least 30 different NPC proteins (Nups) and multiple pore membrane proteins (Poms) is unknown. This project aims to reveal the molecular biogenesis mechanism for the NPC transport apparatus. Our specific aims will each utilize aggressive yeast S. cerevisiae genetics/biochemistry/cell biology-based approaches directly coupled with state-of-the-art microscopy and biochemistry in metazoan cells and the Xenopus egg extract in vitro assembly system. This innovative merger of strengths in multiple model systems is only possible through the full-fledged collaborative efforts of the two PIs. We speculate that de novo assembly is a step-wise process. Our first specific aim will address early assembly events at the NE and chromatin. Using a novel set of yeast NPC assembly mutants and in vitro Xenopus assays, we will identify the essential Nups and assembly factors that are targeted to chromatin, outer and/or inner nuclear membranes. The targeting mechanism and role in assembly will be directly tested. The second aim will focus on the mechanism for fusion of the outer and inner nuclear membranes, i.e. formation of a pore across the NE, and builds on our recent discovery of ER/NE proteins required for NPC assembly and our novel pore fusion assay in Xenopus extracts. We hypothesize that the highly curved pore membranes are formed by the action of Poms, stabilized by transient association of the reticulons, and maintained by the recruitment of a membrane coat formed by the Nup107-160/Nup84 complex. In the third aim, we will analyze the coordination between sequential steps in the biogenesis process. Assembly intermediates will be examined by scanning electron microscopy and purified from yeast mutant cells arrested at distinct steps. We will also pinpoint the order of metazoan Nup recruitment after membrane hole formation by fluorescence imaging of NPC assembly with single pore resolution in real time. Together, these studies will define the membrane fusion machinery and the sequence of self-assembly steps for NPC biogenesis in intact NEs. PUBLIC HEALTH RELEVANCE: Transport of proteins and RNA between the nucleus and cytoplasm is essential for all aspects of normal cell function, and requires large protein machines (nuclear pore complexes) to allow cargo movement. Trafficking is perturbed in some disease states, such as cancer and viral infections, and there are also genetically heritable diseases that are linked to changes in the genes encoding transport factors and nuclear pore complexes. Knowledge of how nuclear pore complexes are assembled and function will be important for finding therapeutic strategies to regulate nuclear transport in disease states and moderate viral proliferation/pathogenesis.