Mitotic roles of the Nuclear Transport Machinery

核运输机械的有丝分裂作用

• 批准号: 10008742
• 负责人: MARY C. DASSO
• 金额: $ 194.13万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10008742
• 关键词: Amyotrophic Lateral Sclerosis; Aneuploidy; Auxins; Binding; Biological; Biological Assay; CRISPR/Cas technology; Cell Line; Cell Nucleus; Cell Survival; Cell physiology; Cells; Chromatin; Chromosome Segregation; Chromosomes; Complex; Cytoplasm; Cytoplasmic Filaments; Cytoskeleton; Data; Defect; Dependence; Development; Enzymes; Filament; GTPase-Activating Proteins; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genome; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Hematologic Neoplasms; Hour; Housekeeping; Human; Individual; Interphase; Kidney Diseases; Kinetochores; MXD1 gene; Maintenance; Mental Retardation; Methods; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modification; Molecular; Neurodegenerative Disorders; Nuclear; Nuclear Envelope; Nuclear Export; Nuclear Import; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Pathology; Pathway interactions; Phenotype; Physiological; Play; Process; Proteins; RNA; RNA Interference; Regulation; Reporting; Role; Solid Neoplasm; Spontaneous abortion; Steroid-resistant idiopathic nephrotic syndrome; Structure; System; Time; Tissues; Work; base; cell growth; chromatin remodeling; chromosome missegregation; experimental study; human disease; in vivo; member; novel; nuclear pore complex protein 96; nuclear pore complex protein p107; nucleocytoplasmic transport; recruit; response; scaffold; trafficking; transcriptome sequencing

Exchange of molecules between the cytoplasm and the nucleus occurs through conduits called nuclear pore complexes (NPCs), which consist of roughly 30 distinct proteins (nucleoporins), forming a central channel with filaments extending into the nucleus and cytoplasm.. Beyond macromolecular trafficking, nucleoporins participate in the control of gene expression via interactions with the genome, as well as in chromatin maintenance and mitotic progression. Their roles in these diverse processes offer a rich variety of possible mechanisms for biological regulation and coordination amongst cellular functions. Recent findings have documented many developmental stage- or tissue-specific phenotypes that result from nucleoporin perturbation, consistent with complex roles that extend beyond simple housekeeping functions. Moreover, human diseases in which nucleoporin function is compromised show remarkably tissue-specific phenotypes, as in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) or in renal diseases like steroid-resistant nephrotic syndromes (SRNS). However, understanding the roles of individual nucleoporins in vertebrate cells is limited because their manipulation by standard methods (e.g., RNAi) has been problematic due to their abundance and their multiple essential roles for cell viability: vertebrate nucleoporin depletion can cause highly pleiotropic phenotypes, many of which may be secondary consequences of extended incubations with sub-physiological nucleoporin levels. To circumvent this problem, we are systematically targeting nucleoporin genes using CRISPR/Cas9 gene editing to create cell lines wherein endogenous nucleoporins have Auxin Inducible Degron (AID) tags, allowing their degradation in a rapid and regulated manner. We are using this approach to analyze the function of individual nucleoporins in a variety of contexts. A major goal of this work is to decipher the specific mechanisms and cellular processes that underlie nucleoporin-based developmental phenotypes and tissue-specific pathologies. We are currently focused on three domains of the NPC. First, Nup153, Tpr, and Nup50 localize to nucleoplasmic filaments, and they are collectively called the basket nucleoporins. The nucleoplasmic filaments have been proposed to serve as a platform for RNA modification and export, as well as for chromatin remodeling. AID-tagged basket nucleoporins localize correctly, are functional within NPCs and are rapidly degraded upon Auxin addition (<2 hours). To assess the role of each nucleoporin, we followed cell growth in the absence and presence of Auxin, as well as nuclear trafficking and the immediate response in gene expression profile (RNA-sequencing). Moreover, we assessed the interdependence of the basket components, and associated with the basket proteins (SENP1, SENP2, MAD1) on each other, on the stability of the assembled nuclear pore, and ability to reform the nuclear pore post mitosis. Our data show that individual basket nucleoporins play distinct roles in nuclear function and gene expression, and that this system provides us the capacity to dissect these roles at a molecular level. Second, the central domain of NPCs consists of three co-axial rings that each display a lattice-like arrangement, and that are called the cytoplasmic ring, inner ring, and nucleoplasmic ring, respectively.The Nup107-160 complex contains nine core nucleoporins (Nup37, Nup85, Seh1, Sec13, Nup96, Nup107, Nup133 and Nup160), with a tenth subunit called ELYS required for chromatin recruitment. The Nup107-160 complex forms the scaffold underlying the cytoplasmic and nuclear rings. The Nup107-160 complex also associates with kinetochores in metazoan mitosis, where it plays a transport-independent role in spindle assembly and chromosome segregation. Earlier efforts at in vivo analysis of individual vertebrate Nup107-160 complex members during interphase and mitosis have been problematic because their abundance and stability makes them difficult to deplete by RNAi: The extended time required for depletion causes progressive defects in both interphase and mitotic functions that can produce adverse secondary consequences. Moreover, the levels of non-targeted subunits decrease during extended RNAi depletion, possibly suggesting that they become unstable when the larger complex is absent. AID-tagged Nup107-160 complex nucleoporins assemble into functional NPCs, and they are degraded rapidly (<4 hours) after auxin addition, with minimal impact on the stability of other Nup107-160 complex members. We have assessed the roles of Nup107-160 complex subunits in nuclear trafficking through comparison of nuclear import and export in the absence and presence of auxin. We are now examining how individual complex members contribute to the structural stability of NPCs, and the inter-dependence between subunits for Nup107-160 complex persistence at existing NPCs, as well as for spindle function and post-mitotic NPC assembly. Third, nucleoporins associated with the cytoplasmic filaments (CFs) include RanBP2 (also known as Nup358), Nup214, Nup88 and Aladin. RanBP2 binds the SUMO1-modified form of the Ran GTPase activating protein (RanGAP1-SUMO1), and the SUMO conjugating enzyme Ubc9 in a stable complex (RRSU complex). CFs interact with transport complexes as they enter and exit the nucleus, and CFs interact with components of the microtubule cytoskeleton. During mitosis, some CF components localize on mitotic spindles and play important roles in spindle assembly or function. In particular, the RRSU complex associates to mitotic kinetochores in a Crm1- and Ran-dependent manner, and this recruitment is important for the formation of spindle-kinetochore attachments. Nup214 and Nup88 have likewise been reported to have important mitotic roles. AID-tagged CF nucleoporins correctly local in functional NPCs. Upon auxin treatment, they are rapidly and specifically degraded, allowing us to assay changes in cellular functions. We are particularly investigating nuclear transport, gene regulation, mitotic progression, and post-mitotic nuclear envelope and NPC re-assembly. These experiments collectively indicate that we are now able to assess the function of individual nucleoporins in vital cellular processes during both interphase and mitosis, and to dissect these processes at a molecular level. This offers an excellent opportunity to assess novel mechanisms of c

细胞质和核之间分子的交换是通过称为核孔复合物（NPC）的导管发生的，该导管由大约30种不同的蛋白质（核孔）组成，形成了一个中心通道，丝状通道延伸到核和细胞质中。维护和有丝分裂进展。它们在这些不同的过程中的作用为生物学调节和细胞功能之间的协调提供了许多可能的机制。最近的发现记录了许多由核孔扰动引起的发育阶段或组织特异性表型，与复杂的作用相一致，这些作用超出了简单的管家功能。此外，核孔功能受到损害的人类疾病表现出非常明显的组织特异性表型，例如在神经退行性疾病中，如肌萎缩性侧面硬化症（ALS）或肾脏疾病（如诸如类固醇抗性肾病综合征（SRNS））中的肾脏疾病。但是，了解单个核孔蛋白在脊椎动物细胞中的作用是有限的，因为它们通过标准方法（例如RNAi）进行操作由于它们的丰度及其在细胞活力中的多个基本作用而存在问题：脊椎动物核核蛋白耗竭会导致高度的菌落现象，其中许多可能会导致替代效果。为了解决这个问题，我们使用CRISPR/CAS9基因编辑来系统地靶向核孔蛋白基因，以创建细胞系，其中内源性核苷具有生长素可诱导的Degron（AID）标签，从而可以快速且受调节的方式降解。我们正在使用这种方法来分析各种情况下各个核孔蛋白的功能。这项工作的主要目的是破译基于基于核孔蛋白的发育表型和组织特异性病理的特定机制和细胞过程。目前，我们专注于NPC的三个领域。 首先，NUP153，TPR和NUP50定位于核质细丝，它们被统称为篮子核苷。已经提出了核质细丝作为RNA修饰和导出的平台，以及染色质重塑。辅助标记的篮子核孔正确定位，在NPC中起作用，并在加入生长素（<2小时）上迅速降解。为了评估每个核孔蛋白的作用，我们在不存在和存在生长素以及核运输以及基因表达谱（RNA-Semecting）中的直接反应下跟随细胞生长。此外，我们评估了篮子成分的相互依存关系，并与篮子蛋白（SENP1，SENP2，MAD1）相关，这是根据组装核孔的稳定性以及改革有丝分裂后改革核孔的能力。我们的数据表明，单个篮子核孔在核功能和基因表达中起着不同的作用，并且该系统为我们提供了在分子水平上剖析这些作用的能力。 其次，NPC的中心结构域由三个同轴环组成，每个环分别称为晶格样环，分别称为细胞质环，内环和核质质环。NUP107-160复合物包含九个核心蛋白NUP160），带有染色质募集所需的第十个亚基。 NUP107-160络合物形成了细胞质和核环的基础支架。 NUP107-160复合物还将其与动力学有丝分裂中的动力学相关联，在该分裂中，它在纺锤体组装和染色体隔离中起着独立于运输的作用。在相间和有丝分裂过程中对个体脊椎动物NUP107-160复合构件进行体内分析的早期努力是有问题的，因为它们的丰度和稳定性使得它们难以被RNAi耗尽：耗尽所需的延长时间会导致相互作用和有丝分裂功能的逐渐缺陷，从而产生不利的次要后果。此外，在延长的RNAi耗竭期间，非靶向亚基的水平降低，可能表明当不存在较大的复合物时它们变得不稳定。辅助标签的NUP107-160复合核丁物组装成功能性NPC，加强生长素后，它们迅速降解（<4小时），对其他NUP107-160复合构件的稳定性影响很小。我们已经通过比较了生长素的存在和存在，评估了NUP107-160复合亚基在核贩运中的作用。现在，我们正在研究单个复合构件如何促进NPC的结构稳定性，以及NUP107-160复杂持久性的亚基之间的相互依赖性，以及纺锤体功能和丝质后NPC组装。 第三，与细胞质丝（CFS）相关的核苷包括RANBP2（也称为NUP358），NUP214，NUP88和Aladin。 RANBP2结合了RAN GTPase激活蛋白（Rangap1-Sumo1）的SUMO1修饰形式，并在稳定的复合物（RRSU复合物）中结合了Sumo Conjugating酶UBC9。 CFS在进入并退出核时与传输复合物相互作用，CFS与微管细胞骨架的组件相互作用。在有丝分裂期间，一些CF组件定位于有丝分裂纺锤体，并在纺锤体组件或功能中起重要作用。特别是，RRSU复合物以CRM1和RAN依赖性方式与有丝分裂的动物学相关，并且该募集对于形成纺锤体 - 金属孔附着很重要。据报道，NUP214和NUP88具有重要的有丝分裂作用。在功能性NPC中正确局部局部AID标记的CF核孔。生长素治疗后，它们会迅速和特异性降解，从而使我们能够测定细胞功能的变化。我们特别研究核转运，基因调节，有丝分裂进展以及有丝分裂后的核包膜和NPC重新组装。 这些实验共同表明，我们现在能够评估单个核孔蛋白在相间和有丝分裂过程中的重要细胞过程中的功能，并在分子水平上剖析这些过程。这提供了评估C的新机制的绝佳机会