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

使用组织特异性，在衰老和应激过程中核孔蛋白在肠核孔复合物的结构、组织和功能中的作用

• 批准号: 2836240
• 金额: --
• 依托单位: Durham University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2836240
• 关键词: Role; nucleoporins; structure; organisation; function

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

核孔复合体(NPC)是一种核质门控通道，可实现核间和胞质间的通讯。它们在物理上连接到细胞骨架和核骨架，并与Sun-Nesprin复合体一起，将细胞核与细胞质连接到细胞外基质。NPC由多个拷贝的~30个核孔蛋白组成。“支架”核孔素“寿命极长”，没有有丝分裂后的周转(D‘Angelo等人，2009年)。然而，它们在衰老/应激过程中丢失和损坏，降低了NPC的功能。利用内源性GFP标记的线虫核孔蛋白菌株，我们观察到核孔蛋白水平的组织特异性差异，提示了特定的作用：例如，ceNup35在肠道和咽部高表达，而MEL-28不表达。我们研究了核孔蛋白在NPC结构和运输中的作用，NPC如何与细胞/核骨架结合，以及细胞骨架在线虫肠道应激敏感性中的作用。线虫提供了一个很好的模型来研究按时间顺序(而不是复制的)衰老和压力对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个肠道细胞都是有丝分裂后的，它们直接暴露在摄入的应激剂中，并且已知肠道参与了衰老/应激。线虫的生命周期很短(所以复杂的可量化的老化实验是可行的)。它是透明的，所以高分辨率荧光成像是直截了当的，几乎没有细胞之间(或细胞周期)的差异，这是体外细胞培养的问题。体积小使得在电子显微镜水平上对整个肠道进行三维重建是可行的。我们的目的是验证这一假设，即在衰老/应激期间NPC的扰动会导致肠道细胞的结构和功能紊乱，从而导致功能障碍。为了测试这一点，我们将确定线虫肠道内的NPC和细胞/核骨骼相互作用是如何随着衰老/应激而改变的。然后，我们将使用RNAi，利用免费可用的资源，测试特定核孔素的扰动如何导致肠道应激/衰老表型。然而，RNAi可能会导致错误组装的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降解系统，或使用可诱导绿色荧光蛋白融合蛋白降解的特定目标肠道(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(扫描电子显微镜)和三维体积EM)来确定细胞/组织结构和NPC结构，并使用功能分析来确定核运输和NPC通透性。利用透射电子显微镜，我们将分析肠道细胞功能(例如，内细胞芽/囊泡形成和微绒毛结构的定量)。重要的是，将使用新的图像分析工具深入分析鼻咽癌的分布和细胞骨架组织。该项目涉及以成像/图像分析为中心的技术。我们的主管/顾问团队拥有所有必要的专业知识。学生将学习：最新的EM方法(高压冷冻/冷冻替代(HPF/FS)、连续切片、透射电子显微镜、三维重建、扫描电子显微镜)；共聚焦显微镜、实时成像和超分辨率技术；普通和蠕虫特有的分子方法