Defining the mechanisms of nuclear pore complex assembly in fission yeast

项目总结 核孔复合体(NPC)横跨内、外核膜，允许调节 大分子在核膜上的运输。此外，NPC有重要的交通工具 独立职能，包括影响核包膜动力学和完整性，有助于 染色体的组织和调控基因的表达。NPC的结构和功能的许多特点是 在真核生物中是保守的；然而，NPC的数量、分布、组成和功能可以改变 在开发过程中和对环境信号的反应中戏剧性地。此外，增加了NPC密度 已经在包括癌症在内的人类疾病中观察到。在许多生物体中的研究已经确定了 Ndc1作为鼻咽癌组装和插入所需的关键因子 核包膜。然而，鼻咽癌插入的确切机制仍不清楚。另外， 试图剖析酵母中Ndc1和其他假定的插入因子在npc上的作用是复杂的。 通过它们在插入酵母纺锤体极体中的双重功能。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分裂酵母相互作用的蛋白质 Ndc1正交系，Cut11。利用高通量膜酵母-双杂交筛选法，我们鉴定了新的Cut11 与脊椎动物中保守的脂代谢和膜组织有关的相互作用蛋白 并确定了这些相互作用是如何受到Cut11突变的调制的。我们还开发了3D 结构照明显微镜和图像分析工具，使我们能够可视化和量化体内的NPC。 重要的是，这种方法允许确定NPC的组成，同时保持单一的NPC决议，以及 揭示了纺锤极体附近有一个NPC成分不均匀的区域。我们将利用这一点 强大的成像平台来确定新发现的Cut11相互作用蛋白是否定位于NPC 并具有鼻咽癌组装和插入的功能。我们还将研究分子机制， 调控保守的核膜蛋白Tts1在鼻咽癌中的定位并阐明其作用 装配和分配。最后，我们将定义主轴极体附近区域的NPC的组成 并确定如何建立和维护这一专门的NPC池。这些研究将扩大我们的 了解鼻咽癌组装是如何调控的，将为研究新的蛋白质提供有价值的见解 在从酵母到哺乳动物的NPC组装中具有潜在的保守功能。

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.