Role of nucleoporins in the structure, organisation, and function of intestinal nuclear pore complexes, during ageing and stress, using tissue specifi

核孔复合物（NPC）是核质门控通道，使核和细胞质区室之间的通信。它们与细胞骨架和核骨架物理连接，并与SUN-Nesprin复合物一起将细胞核连接到细胞质和细胞外基质。NPC由~30个核孔蛋白的多个拷贝组成。“支架”核孔蛋白“寿命极长”，无有丝分裂后更新（D 'Angelo et al 2009）。然而，它们在老化/压力期间丢失和损坏，降低NPC功能。使用内源性GFP标记的核孔蛋白C.我们研究了核孔蛋白在NPC结构和转运中的作用，NPC如何与细胞/核骨架结合，以及细胞骨架在C. elegans应激敏感性中的作用。线虫肠C. elegans提供了一个很好的模型来研究时序性（而不是复制性）衰老和应激对NPC的影响，以及NPC在细胞组织和功能中的作用：在成年人中，所有20个肠细胞都是有丝分裂后的，它们直接暴露于摄入的应激剂，并且已知肠参与衰老/应激。C.线虫的生命周期很短（因此复杂的可量化的衰老实验是可行的）。它是透明的，因此高分辨率荧光成像是直接的，并且几乎没有细胞与细胞（或细胞周期）的变化，这是体外细胞培养的一个问题。小尺寸使得在电子显微镜水平上对整个肠道进行3D重建成为可能。我们的目的是检验这一假设，即衰老/应激期间NPC扰动导致肠细胞结构和功能紊乱，导致功能障碍。为了验证这一点，我们将确定NPC和细胞/核骨架相互作用是如何在C内响应老化/应激而改变的。线虫肠然后，我们将测试特定核孔蛋白的扰动如何导致肠道应激/衰老表型，使用RNAi，免费提供的资源。然而，RNAi可能导致错误组装的NPC。为了更准确地模拟衰老/损伤过程，我们将使用生长素诱导的降解决定子（AID）系统或用诱导的GFP融合蛋白降解特异性靶向肠道（使用我们的合作者提供的菌株（doi.org/10.1101/2021.12.21.473632））。我们将使用结构分析（超分辨率光学显微镜，透射和扫描电子显微镜（SEM，TEM）和3D体积EM）来确定细胞/组织和NPC结构，以及核转运和NPC渗透性的功能测定。使用TEM，我们将测定肠细胞功能（例如内吞芽/囊泡形成和微绒毛结构的定量）。重要的是，NPC的分布和细胞骨架组织将使用新的图像分析工具进行深入分析。该项目涉及围绕成像/图像分析的技术。我们的管理人员/顾问团队拥有所有必要的专业知识。学生将学习：最先进的EM方法（高压冷冻/冷冻置换（HPF/FS）、连续切片、TEM、3D重建、SEM）;共聚焦显微镜、实时成像和超分辨率技术;一般和蠕虫特异性分子方法

Nuclear pore complexes (NPCs) are nucleo-cytoplasmic gated channels that enable communication between the nuclear and cytoplasmic compartments. They physically link to the cytoskeleton and nucleoskeleton, and together with SUN-Nesprin complexes, connect the nucleus to the cytoplasm to the extra-cellular matrix. The NPC consists of multiple copies of ~30 nucleoporins. "Scaffold" nucleoporins are "extremely long-lived" with no post-mitotic turnover (D'Angelo et al 2009). They are however lost and damaged during aging/stress, degrading NPC function. Using endogenously GFP-tagged nucleoporin strains of C. elegans, we observed tissue-specific variation in nucleoporin levels, suggesting specific roles: e.g. ceNup35 is highly expressed in intestine versus pharynx, whereas MEL-28 is not.We study roles of nucleoporins in NPC structure and transport, how NPCs associate with cyto/nucleoskeletons, and the roles of the cytoskeleton in stress-sensitivity of C. elegans intestine. C. elegans provides an excellent model to study effects of chronological (rather than replicative) aging and stress on NPCs, and NPC roles in cellular organisation and function: in adults, all 20 intestinal cells are post-mitotic, they are directly exposed to ingested stress agents, and the intestine is known to be involved in aging/stress. C. elegans has a short life cycle (so complex quantifiable aging experiments are feasible). It is transparent, so high-resolution fluorescence imaging is straightforward and there is little cell to cell (or cell cycle) variation, a problem with in vitro cell cultures. Small size makes 3D reconstruction of the whole intestine at the electron microscope level feasible. Our aim is to test the hypothesis that NPC perturbations during aging/stress cause structural and functional dis-organisation of intestinal cells leading to disfunction. To test this, we will determine how NPCs and cyto/nucleoskeletal interactions are altered in response to aging/stress within C. elegans intestine. We will then test how perturbation of specific nucleoporins causes intestinal stress/aging phenotypes, using RNAi, with freely available resources. RNAi may however lead to mis-assembled NPCs. To more accurately mimic aging/damage processes, we will use the auxin inducible degron (AID) system or specifically target the intestine with inducible GFP-fusion protein degradation (using strains provided by our collaborator (doi.org/10.1101/2021.12.21.473632)). We will use structural analyses (super resolution light microscopy, transmission and scanning electron microscopy (SEM, TEM) and 3D volume EM) to determine cellular/tissue organisation and NPC structure, and functional assays for nuclear transport and NPC permeability. Using TEM, we will assay intestinal cell function (e.g. quantification of endocytic bud/vesicle formation and microvilli structure). Importantly, NPC distribution and cytoskeleton organisation will be analysed in-depth using novel image analysis tools.The project involves techniques centred around imaging/image analysis. Our team of supervisors/advisors have all necessary expertise. Student will learn: state-of-the-art EM methods (high pressure freezing/freeze substitution (HPF/FS), serial sectioning, TEM, 3D reconstruction, SEM); confocal microscopy, live imaging and super resolution techniques; general and worm-specific molecular methods