Defining the mechanisms of nuclear pore complex assembly in fission yeast

定义裂殖酵母核孔复合体组装的机制

• 批准号: 9909195
• 负责人: Joseph M Varberg
• 金额: $ 6.53万
• 依托单位: STOWERS INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-05 至 2021-09-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9909195
• 关键词: 3-Dimensional; Active Sites; Address; Affect; Alleles; Animal Model; Binding; Carrier Proteins; Cells; Chromosome Structures; Color; Complex; Development; Enzymes; Estrogen receptor positive; Eukaryota; Exclusion; Fission Yeast; Gene Expression; Genetic; Genetic Recombination; Genetic Techniques; Goals; Heterogeneity; Human; Image; Image Analysis; Imaging Techniques; Interphase; Lighting; Lipids; Malignant Neoplasms; Mammals; Membrane; Membrane Lipids; Membrane Proteins; Methods; Microscopy; Mitosis; Modeling; Molecular; Monitor; Mutation; Nature; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Outer Membrane; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Organism; Orthologous Gene; Process; Proteins; Reporting; Research Personnel; Resolution; Role; Saccharomycetales; Shapes; Signal Transduction; Structure; System; Testing; Training; Two-Hybrid System Techniques; Vertebrates; Work; Yeasts; base; career; density; env Gene Products; genetic approach; human disease; imaging approach; imaging genetics; imaging platform; in vivo; innovation; insight; lipid metabolism; macromolecule; microscopic imaging; novel; nucleocytoplasmic transport; particle; quantitative imaging; response; spindle pole body; telophase; three dimensional structure; tool; yeast genetics; yeast two hybrid system

PROJECT SUMMARY Nuclear pore complexes (NPCs) span the inner and outer nuclear membranes and allow for the regulated transport of macromolecules across the nuclear envelope. In addition, NPCs have important transport independent functions, including influencing nuclear envelope dynamics and integrity, contributing to chromosomal organization and regulating gene expression. Many features of NPC structure and function are conserved throughout eukaryotes; however, NPC number, distribution, composition and function can change dramatically during development and in response to environmental signals. Additionally, increased NPC density has been observed in human diseases including cancer. Work in numerous organisms has identified the conserved transmembrane nucleoporin Ndc1 as a key factor required for NPC assembly and insertion into the nuclear envelope. However, the exact mechanism by which NPC insertion occurs remains unclear. Additionally, attempts to dissect the role of Ndc1 and other putative insertion factors at NPCs in yeast have been complicated by their dual functions in the insertion of the yeast spindle pole body. To overcome these issues, we have developed innovative genetic and imaging approaches to characterize proteins that interact with the fission yeast Ndc1 ortholog, Cut11. Using high-throughput membrane yeast-two hybrid screens, we identified novel Cut11 interacting proteins involved in lipid metabolism and membrane organization that are conserved in vertebrates and have determined how these interactions are modulated by Cut11 mutations. We have also developed 3D structured illumination microscopy and image analysis tools to allow us to visualize and quantify NPCs in vivo. Importantly, this approach allows for determining NPC composition while maintaining single NPC resolution, and has revealed a region with heterogenous NPC composition near the spindle pole body. We will utilize this powerful imaging platform to determine whether the newly identified Cut11 interacting proteins localize to NPCs and have a function in NPC assembly and insertion. We will also examine the molecular mechanisms that regulate the localization of the conserved nuclear envelope protein Tts1 to the NPCs and clarify its role in NPC assembly and distribution. Last, we will define the composition of NPCs in the region near the spindle pole body and determine how this specialized pool of NPCs is established and maintained. These studies will expand our understanding of how NPC assembly is regulated and will provide valuable insight into novel proteins with potentially conserved functions in NPC assembly from yeast to mammals.

项目概要 核孔复合物 (NPC) 跨越内核膜和外核膜，并允许调节 大分子穿过核膜的运输。此外，NPC还有重要的交通工具 独立的功能，包括影响核膜动力学和完整性，有助于 染色体组织和调节基因表达。 NPC结构和功能的许多特点是 在真核生物中保守；然而，NPC的数量、分布、组成和功能可能会改变 在发育过程中和对环境信号的响应中显着。此外，NPC 密度增加 已在包括癌症在内的人类疾病中观察到。在许多生物体中的工作已经确定 保守的跨膜核孔蛋白 Ndc1 作为 NPC 组装和插入所需的关键因子 核膜。然而，NPC 插入发生的确切机制仍不清楚。此外， 剖析 Ndc1 和其他假定的酵母 NPC 插入因子的作用的尝试很复杂 通过它们插入酵母纺锤体极体的双重功能。为了克服这些问题，我们有 开发了创新的遗传和成像方法来表征与裂殖酵母相互作用的蛋白质 Ndc1 直系同源物，Cut11。使用高通量膜酵母-两种杂交筛选，我们鉴定了新型 Cut11 参与脊椎动物保守的脂质代谢和膜组织的相互作用蛋白 并确定了 Cut11 突变如何调节这些相互作用。我们还开发了3D 结构照明显微镜和图像分析工具使我们能够在体内可视化和量化 NPC。 重要的是，这种方法允许确定 NPC 组成，同时保持单个 NPC 分辨率，并且 在纺锤体极体附近发现了一个具有异质 NPC 组成的区域。我们将利用这个 强大的成像平台，可确定新鉴定的 Cut11 相互作用蛋白是否定位于 NPC 并具有NPC组装和插入的功能。我们还将研究分子机制 调节保守核膜蛋白 Tts1 在 NPC 中的定位并阐明其在 NPC 中的作用 组装和分发。最后定义纺锤体附近区域的NPC组成 并确定如何建立和维护这个专门的 NPC 库。这些研究将扩大我们的 了解 NPC 组装如何调节，将为研究新型蛋白质提供有价值的见解 从酵母到哺乳动物的 NPC 组装中潜在的保守功能。