Establishing a blueprint for nuclear pore complex assembly

建立核孔复合体组装蓝图

• 批准号: 8690921
• 负责人: Charles Patrick Lusk
• 金额: $ 31.56万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8690921
• 关键词: Alzheimer' s Disease; Animal Model; Automobile Driving; Biogenesis; Cell Nucleus; Cell membrane; Cell physiology; Coupled; Cytoplasm; Data; Data Set; Development; Dimerization; Disease; Drug Targeting; Eukaryota; Event; Fluorescent Probes; Future; Generations; Genes; Genetic; Goals; Health; Heart Diseases; Home environment; Human; In Vitro; Individual; Interphase; Left; Light; Liposomes; Malignant Neoplasms; Membrane; Membrane Fusion; Membrane Proteins; Mitotic; Molecular; Mutation; Neurodegenerative Disorders; Nuclear; Nuclear Envelope; Nuclear Outer Membrane; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nucleoplasm; Parkinson Disease; Pathology; Pathway interactions; Permeability; Process; Proteins; Saccharomyces cerevisiae; Saccharomycetales; Series; Sirolimus; Stream; Syndrome; System; Tertiary Protein Structure; Testing; Therapeutic; Time; TimeLine; Tissues; Translational Research; Virus Diseases; Work; Yeasts; base; design; genetic resource; in vivo; insight; macromolecule; nucleocytoplasmic transport; overexpression; programs; public health relevance; reconstitution; research study; success

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.

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）分子双向交换的唯一网关。从人类健康的角度来看，NPC 的几种成分（核孔蛋白/nups）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，病毒经常调节nup功能。这些病理学的广泛范围表明 NPC 影响广泛的基本细胞过程，尽管其机制尚不清楚。此外，在发育和疾病背景下，一些nup基因被上调或下调，并且NPC数量被改变。因此，更好地了解有助于 NPC 功能的机制可能会在未来揭示转化药物靶点。虽然作为一个领域，我们对控制核运输的基本机制有很好的了解，但剩下的一个主要挑战是确定从头 NPC 组装的机制。具体来说，尚不清楚约 30 个个体 nups 如何在空间和时间上协调组装以形成约 50 个 MD NPC。此外，单个核的组装与膜曲率的产生同时发生，膜曲率导致内核膜和外核膜紧密并置并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，也不清楚融合机制。在这项提案中，我们旨在解决理解全国人大会议的两个关键挑战。首先，我们提出了一种实验策略，旨在阐明间期期间核在 NE 处组装的顺序。我们将通过利用酿酒酵母的遗传工具包来实现这一目标，以生成一个系统，在该系统中，我们可以快速、特异地一次灭活一个新合成的 nups，而成熟的 NPC 不受影响。每个目标nup失活后，我们将综合检查其他nup的分布情况，以按组装顺序分配上游或下游关系。整合该数据集，我们将系统地定义装配过程中的步骤。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 中孔形成的蛋白质。使用一系列体内和体外方法，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并引导其曲率生成域。通过这种方式，我们将为孔形成的分子机制提供重要的新线索，并直接测试膜曲率如何影响 NPC 组装。该提案中概述的实验将为所有真核生物普遍存在的 NPC 生物发生的基本过程提供急需的机制见解。