Regulation of Nuclear Pore Complex Assembly

描述（由申请人提供）：核孔复合物（npc）是进入和离开细胞核的唯一场所。这包括小分子的扩散和大蛋白质和RNA的选择性、主动运输。适当的鼻咽癌生物发生对细胞分裂、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策略的关键，例如在肿瘤发生中。然而，npc重新插入完整核膜（NE）的分子途径在很大程度上是不明确的。该领域最突出的问题涉及描述核孔形成是如何执行和调节的。至少30种不同的NPC蛋白（Nups）和多个孔膜蛋白（Poms）之间协调相互作用的途径尚不清楚。本项目旨在揭示NPC转运装置的分子生物学机制。我们的具体目标是利用侵略性酵母酵母遗传学/生物化学/细胞生物学为基础的方法，直接结合最先进的显微镜和生物化学在后生动物细胞和爪蟾卵提取物的体外组装系统。在多个模型系统中，这种创新的优势合并只有通过两个pi的全面合作才能实现。我们推测，从头组装是一个循序渐进的过程。我们的第一个具体目标是解决NE和染色质的早期组装事件。利用一组新的酵母NPC组装突变体和体外Xenopus试验，我们将确定针对染色质，外核膜和/或内膜的基本Nups和组装因子。靶向机制及其在装配中的作用将直接得到检验。第二个目标将集中于外核膜和内核膜融合的机制，即在NE上形成一个孔，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进行的新型孔融合试验的基础上。我们假设高度弯曲的孔膜是由Poms的作用形成的，通过网状结构的短暂结合来稳定，并通过由Nup107-160/Nup84复合物形成的膜涂层的募集来维持。在第三个目标中，我们将分析生物发生过程中顺序步骤之间的协调。组装中间体将通过扫描电子显微镜检查，并从不同步骤捕获的酵母突变细胞中纯化。我们还将通过单孔分辨率的NPC组件实时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NEs中鼻咽癌生物发生的膜融合机制和自组装步骤序列。

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.