Defining the mechanisms of nuclear pore complex assembly in fission yeast

项目摘要 核孔复合物（NPC）跨越核膜内外，并允许调节细胞内的蛋白质。 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， 在整个真核生物中保守;然而，NPC的数量，分布，组成和功能可以改变 在发育过程中以及对环境信号的反应中发生显著变化。此外，NPC密度增加 已经在包括癌症在内的人类疾病中观察到。在许多生物体中的工作已经确定了 保守的跨膜核孔蛋白Ndc 1是NPC组装和插入到细胞膜中所需的关键因子。 核膜然而，NPC插入发生的确切机制仍不清楚。此外，本发明还 试图剖析Ndc 1和其他假定的插入因子在酵母NPC中的作用是复杂的 通过它们在酵母纺锤体极体插入中的双重功能。为了解决这些问题，我们 开发了创新的遗传和成像方法来表征与裂变酵母相互作用的蛋白质 Ndc 1直系同源物，Cut 11。使用高通量膜酵母双杂交筛选，我们确定了新的Cut 11 在脊椎动物中保守的参与脂质代谢和膜组织的相互作用蛋白质 并确定了这些相互作用是如何被Cut 11突变所调节的。我们还开发了3D 结构化照明显微镜和图像分析工具，使我们能够可视化和量化的NPC在体内。 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 揭示了一个区域与异质NPC组成的纺锤体极体附近。我们会利用这个 强大的成像平台，以确定新发现的Cut 11相互作用蛋白是否定位于NPC 并且具有NPC组装和插入的功能。我们还将研究分子机制， 调节保守的核膜蛋白Tts 1在NPC中的定位，阐明其在NPC中的作用 组装和分配。最后，我们将确定在纺锤体附近区域的NPC的组成 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组装是如何调节的，并将提供有价值的洞察新的蛋白质， 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.