NPC Structure in Model Organisms: Transmission Electron Microscopy and Immunogold Labeling Using High-Pressure Freezing/Freeze Substitution of Yeast, Worms, and Plants.
NPC Structure in Model Organisms: Transmission Electron Microscopy and Immunogold Labeling Using High-Pressure Freezing/Freeze Substitution of Yeast, Worms, and Plants.
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模型生物中的 NPC 结构：使用酵母、蠕虫和植物的高压冷冻/冷冻替代的透射电子显微镜和免疫金标记。
DOI：
10.1007/978-1-0716-2337-4_28
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Richardson AC
中科院分区：
文献类型：
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作者：
Richardson AC
核孔复合体（NPC）是嵌入核膜内的大型精细结构，与细胞骨架、核骨架和染色质密切相关。许多不同的货物通过其中央通道和沿膜在其外围。NPC在每个细胞周期中全部或部分地拆卸和重新组装。在有丝分裂后的细胞中，它由超稳定和高动态蛋白质的组合组成。由于它的大小、动力学、异质性和整合性，不可能通过任何一种甚至几种方法来了解它的结构和分子功能。几十年来，直到今天，薄片透射电子显微镜（TEM）一直是了解NPC及其关联，动力学和运输中的作用的核心工具，因为它可以独特地回答有关细胞背景下精细结构细节的问题。使用免疫金标记的特定成分也可以在超微结构背景下识别。酿酒糖酵素等模式生物也是鼻咽癌研究的核心，但尚未广泛用于结构工作。这是因为细胞壁在结构保存和透射电镜处理方面存在困难。近年来，高压冷冻和冷冻替代技术克服了这些问题，开辟了免疫金标记与详细结构分析相结合的方法。其他模式生物，如秀丽隐杆线虫和植物拟南芥，由于类似的原因没有得到充分利用，但我们在这里提出了类似的解决方案。由于初始固定的即时性，使用这些方法也有许多优点，适用于哺乳动物系统，以捕捉快速过程，如核运输和动态膜的保存。
The nuclear pore complex (NPC) is a large elaborate structure embedded within the nuclear envelope, and intimately linked to the cytoskeleton, nucleoskeleton, and chromatin. Many different cargoes pass through its central channel and along the membrane at its periphery. The NPC is dismantled and reassembly, fully or partially, every cell cycle. In post-mitotic cells it consists of a combination of hyper-stable and highly dynamic proteins. Because of its size, dynamics, heterogeneity and integration, it is not possible to understand its structure and molecular function by any one, or even several, methods. For decades, and to this day, thin section transmission electron microscopy (TEM) has been a central tool for understanding the NPC, its associations, dynamics and role in transport as it can uniquely answer questions concerning fine structural detail within a cellular context. Using immunogold labeling specific components can also be identified within the ultrastructural context. Model organisms such asSaccharomyces cerevisiaeare also central to NPC studies but have not been used extensively in structural work. This is because the cell wall presents difficulties with structural preservation and processing for TEM. In recent years, high-pressure freezing and freeze substitution have overcome these problems, as well as opened up methods to combine immunogold labeling with detailed structural analysis. Other model organisms such as the wormCaenorhabditis elegansand the plantArabidopsis thalianahave been underused for similar reasons, but with similar solutions, which we present here. There are also many advantages to using these methods, adapted for use in mammalian systems, due to the instant nature of the initial fixation, to capture rapid processes such as nuclear transport, and preservation of dynamic membranes.